N-[1, (1-1) -dialkyloxy] - and N- [1, (1-1) -dialkenyloxy]- alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor

ABSTRACT

This invention relates to compounds of the formula                    
     or an optical isomer thereof wherein R 1  and R 2  are the same or different and are an alkyl or alkenyl group of 6 to 24 carbon atoms; R 3 , R 4  and R 5  are the same or different and are alkyl of 1 to 8 carbon atoms, aryl, aralkyl of 7 to 11 carbon atoms, or when two or three of R 3 , R 4 , and R 5  are taken together to form quinuclidino, piperidino, pyrrolidino, or morpholino; n is 1 to 8; and X is a pharmaceutically acceptable anion.

A. RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/237,807, filed May 5,1994, now U.S. Pat. No. 5,622,712; which is a division of applicationSer. No. 08/015,738, filed Feb. 10, 1993, now U.S. Pat. No. 5,366,737;which is a division of application Ser. No. 07/614,412, filed Nov. 16,1990, now U.S. Pat. No. 5,208,036; which is a division of applicationSer. No. 07/524,257, filed May 15, 1990, now U.S. Pat. No. 5,049,386;which is a division of application Ser. No. 07/428,815, filed Oct. 27,1989, now U.S. Pat. No. 4,946,787; which is a division of applicationSer. No. 07/114,809, filed Oct. 29, 1987, now U.S. Pat. No. 4,897,355;which is a continuation-in-part of application Ser. No. 06/877,916,filed Jun. 24, 1986, now abandoned; which is a continuation-in-part ofapplication Ser. No. 06/689,407, filed Jan. 7, 1985, now abandoned.

I. BACKGROUND OF THE INVENTION

B. Field of the Invention

This invention relates to lipophilic cationic compounds and several oftheir uses. The invention also relates to a novel DNA transfectionmethod, in which the compounds of this invention can be used.

C. Related Art

Liposomes are microscopic vesicles consisting of concentric lipidbilayers. Structurally, liposomes range in size and shape from longtubes to spheres, with dimensions from a few hundred Angströms tofractions of a millimeter. Regardless of the overall shape, the bilayersare generally organized as closed concentric lamellae, with an aqueouslayer separating each lamella from its neighbor. Vesicle size normallyfalls in a range of between about 20 and about 30,000 nm in diameter.The liquid film between lamellae is usually between about 3 and 10 nm.

Typically, liposomes can be divided into three categories based on theiroverall size and the nature of the lamellar structure. The threeclassifications, as developed by the New York Academy Sciences Meeting,“Liposomes and Their Use in Biology and Medicine,” of December 1977, aremulti-lamellar vesicles (MLV's), small uni-lamellar vesicles (SUV's) andlarge uni-lamellar vesicles (LUV's).

SUV's range in diameter from approximately 20 to 50 nm and consist of asingle lipid bilayer surrounding an aqueous compartment. Unilamellarvesicles can also be prepared in sizes from about 50 nm to 600 nm indiameter. While unilamellar are single compartmental vesicles of fairlyuniform size, MLV's vary greatly in size up to 10,000 nm, orthereabouts, are multi-compartmental in their structure and contain morethan one bilayer. LUV liposomes are so named because of their largediameter which ranges from about 600 nm to 30,000 nm; they can containmore than one bilayer.

Liposomes may be prepared by a number of methods not all of whichproduce the three different types of liposomes. For example, ultrasonicdispersion by means of immersing a metal probe directly into asuspension of MLV's is a common way for preparing SUV's.

Preparing liposomes of the MLV class usually involves dissolving thelipids in an appropriate organic solvent and then removing the solventunder a gas or air stream. This leaves behind a thin film of dry lipidon the surface of the container. An aqueous solution is then introducedinto the container with shaking in order to free lipid material from thesides of the container. This process disperses the lipid, causing it toform into lipid aggregates or liposomes.

Liposomes of the LUV variety may be made by slow hydration of a thinlayer of lipid with distilled water or an aqueous solution of some sort.

Alternatively, liposomes may be prepared by lyophilization. This processcomprises drying a solution of lipids to a film under a stream ofnitrogen. This film is then dissolved in a volatile solvent, frozen, andplaced on a lyophilization apparatus to remove the solvent. To prepare apharmaceutical formulation containing a drug, a solution of the drug isadded to the lyophilized lipids, whereupon liposomes are formed.

A variety of methods for preparing various liposome forms have beendescribed in the periodical and patent literature. For specific reviewsand information on liposome formulations, reference is made to reviewsby Pagano and Weinstein (Ann. Rev. Biophysic. Bioeng., 7, 435-68 (1978))and Szoka and Papahadjopoulos (Ann. Rev. Biophysic. Bioeng., 9, 467-508(1980)) and additionally to a number of patents, for example, U.S. Pat.Nos. 4,229,360; 4,224,179; 4,241,046; 4,078,052; and 4,235,871.

Thus, in the broadest terms, liposomes are prepared from one or morelipids. Though it has been thought that any type of lipid could be usedin liposomes, e.g. cationic, neutral or anionic lipids, experience withpositively charged liposomes has indicated several problems which havenot been fully addressed to date. The amines which have to date beenemployed in preparing cationic liposomes have either not beensufficiently chemically stable to allow for the storage of the vesicleitself (short shelf life) or the structure of the amines has been suchthat they can be leached out of the liposome bilayer. One such amine,stearylamine, has toxicity concerns which limit its use as a componentof liposomes in a pharmaceutical formulation. Another amine, dimethyldioctadecyl ammonium bromide, lacks the appropriate molecular geometryfor optimum formation of the bilayers that comprise the liposomestructure.

Various biological substances have been encapsulated into liposomes bycontacting a lipid with the matter to be encapsulated and then formingthe liposomes as described above. A drawback of this methodology,commonly acknowledged by those familiar with the art, is that thefraction of material encapsulated into the liposome structure isgenerally less than 50%, usually less than 20%, often necessitating anextra step to remove unencapsulated material. An additional problem,related to the above, is that after removal of unencapsulated material,the encapsulated material can leak out of the liposome. This secondissue represents a substantial stability problem to which much attentionhas been addressed in the art.

Liposomes have been used to introduce DNA into cells. More specifically,various DNA transfection methodologies have been used, includingmicroinjection, protoplast fusion, liposome fusion, calcium phosphateprecipitation, electroporation and retroviruses. All of these methodssuffer from some significant drawbacks: they tend to be too inefficient,too toxic, too complicated or too tedious to be conveniently andeffectively adapted to biological and/or therapeutic protocols on alarge scale. For instance, the calcium phosphate precipitation methodcan successfully transfect only about 1 in 10⁷ to 1 in 10⁴ cells; thisfrequency is too low to be applied to current biological and/ortherapeutic protocols. Microinjection is efficient but not practical forlarge numbers of cells or for large numbers of patients. Protoplastfusion is more efficient than the calcium phosphate method but thepropylene glycol that is required is toxic to the cells. Electroporationis more efficient then calcium phosphate but requires a specialapparatus. Retroviruses are sufficiently efficient but the introductionof viruses into the patient leads to concerns about infection andcancer. Liposomes have been used before but the published protocols havenot been shown to be any more efficient than calcium phosphate. The mostdesirable transfection method would involve one that gives very highefficiency without the introduction of any toxic or infectioussubstances and be simple to perform without a sophisticated apparatus.The method that we describe satisfies all of these criteria.

II. SUMMARY OF THE INVENTION

According to a first aspect of the invention, the compounds of thisinvention are illustrated by Formula (I):

or an optical isomer thereof, wherein R¹ and R² are independently analkyl, alkenyl, or alkynyl group of 6 to 24 carbon atoms; R³, R⁴ and R⁵are independently hydrogen, alkyl of 1 to 8 carbon atoms, aryl oraralkyl of 6 to 11 carbon atoms; alternatively two or three of R³, R⁴and R⁵ are combined with the positively charged nitrogen atom to form acyclic structure having from 5 to 8 atoms, where, in addition to thepositively charged nitrogen atom, the atoms in the structure are carbonatoms and can include one oxygen, nitrogen or sulfur atom; n is 1 to 8;and X is an anion.

According to other aspects of the invention, liposome and pharmaceuticalformulations are claimed: said liposome formulations comprising up tolot by weight of a biologically active substance, 1% to 20% by weight ofa lipid component comprising a compound of Formula I in a quantity offrom about It to loot by weight, and an aqueous solution in a quantitysufficient to make 100% by volume; and said pharmaceutical formulationscomprising a therapeutically effective amount of a drug, an optionalpharmaceutically acceptable excipient, and a lipid component comprisinga compound of Formula I in a quantity of from about 1% to 100% byweight.

According to another aspect of the invention, a polyanion-lipid complex,formed from a compound of formula I and a polyanion, is claimed.

According to yet another aspect of the invention, a method is claimedfor forming a polyanion-lipid complex, said method comprising the stepsof contacting a liposomal composition prepared from a positively chargedliposome-forming lipid with a negatively charged polyanion.

According to still another aspect of the invention, a positively-chargedpolynucelotide-liposome complex is claimed, comprising a lipid ofFormula I and a polynucleotide.

According to a further aspect of the invention, a method is claimed forpreparing a positively-charged polynucleotide-lipid complex. The methodcomprises the steps of contacting a positively charged liposome madefrom a lipid of Formula I with a polyanion.

According to yet another aspect of the invention, a method is claimedfor introducing a polyanion into a cell. The method comprises forming aliposome from a lipid of Formula I, contacting the liposome with apolyanion to form a positively-charged polyanion-liposome complex, andincubating the complex with a cell.

According to still another aspect of the invention, a method is claimedfor intracellularly delivering a biologically active substance, whichmethod comprises forming a liposome comprising a lipid of Formula I anda biologically active substance, and incubating the liposome with a cellculture.

According to a further aspect of the invention, an antigenic formulationis claimed, comprising an antigen and a compound of Formula I.

According to a still further aspect of the invention, a method isclaimed for the transdermal, topical or ocular delivery of a drug. Themethod comprises the steps of forming a liposome comprising a compoundof Formula I and the drug; and applying the liposome to the skin ormucous membranes of a human or animal subject.

According to another aspect of the invention, double coated liposomecomplexes are claimed, comprising a polyanion, a lipid of Formula I, anda negatively charged co-lipid.

According to a still further aspect of the invention, a method isclaimed for making said double-coated complexes, comprising forming aliposome from a lipid of Formula I; contacting it with a polyanion; andcontacting the resulting complex with an excess of negatively-chargedlipid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bar graph of transfection efficiency as a function of DNAconcentration.

FIG. 1B is a bar graph of transfection efficiency as a function of lipidconcentration.

FIG. 1C is a bar graph of transfection efficiency as a function ofincubation time.

III. DETAILED DESCRIPTION OF THE INVENTION

Several advantages flow from the compounds and methods of the presentinvention. One of the advantages of the methods and materials disclosedherein is that they permit up to 100% entrapment of polyanionicsubstances by an exceedingly convenient and practical protocol. Anotheradvantage of the liposome compositions disclosed herein is that they arenot subject to instability due to leakage of the entrapped polyanionicsubstance. Still another advantage is that the convenient and practicalmethodology disclosed herein yields compositions of matter with uniqueproperties enabling entry of the entrapped polyanionic substance, suchas DNA, into living cells. This property of the resultinglipid/polyanion complex enables the expression of biological activitiesto extents not previously seen in these cells. And still further, thismethodology leads to results that have not been obtained withconventional liposomes.

The positively charged pharmaceutical formulations, particularlyliposomes, of this invention are pharmaceutically advantageous: thepresentation of positively charged materials to the negatively chargedcell surface results in better uptake of the pharmaceutical materials bythe cells.

The unique advantages of the technology disclosed herein are of twotypes. First, the compounds of Formula I represent novel positivelycharged liposome forming lipids, which can be used for the formation ofpositively charged liposomes in which drugs or other materials can beencapsulated in the conventional manner. The uniqueness of this aspectof the invention depends on the chemical structure of the compounds ofFormula I. The principal advantages of this structure derive from thegeometry of the two parallel aliphatic chains, the overall positivecharge of the molecule itself, and the chemical stability of the etherlinkages. The geometry of the two aliphatic chains enables theorganization by the compounds of Formula I into stable bilayerstructures. These bilayers comprise the overall structure of theliposome itself. The positive charge on the molecules of Formula Iprovides the resulting liposome with an overall positive charge,resulting in a net positively charged liposome. The ether linkage of thealiphatic chains provides the chemical stability important for the typeof chemical structure synthesized and for the type of applicationsdescribed herein. Both hydrophobic and hydrophilic biologically activesubstances can be incorporated into the resulting liposomes usingconventional liposome technology commonly known by those familiar withthe art. The resulting liposomes produced are better than those producedwith other commonly available materials, because the compounds ofFormula I have a geometry more compatible with the formation ofbilayers, leading to a liposome with greater physical stability.

Thus, compounds of Formula I do not suffer from the drawbacks of aminesemployed in liposomes before this invention. The ether linkage of thecompounds of Formula I is highly stable in liposomes. Additionally, thecompounds of Formula I are not leached out of nor do they otherwisemigrate out of the liposome matrix as do stearyl amines and otheramines. Moreover, concerns of toxicity are significantly reduced withthe compounds of Formula I. Still further, the parallel geometry of thealiphatic chains in the preferred embodiments of the compounds ofFormula I overcomes problems with bilayer compatibility that are commonto molecules such as dioctadecyldimethyl ammonium bromide.

The second unique advantage of the technology disclosed herein isderived from the novel method for incorporating polyanionic biologicallyactive substances into a liposome complex. This complex is composed ofpositively charged liposomes prepared from compounds of Formula I orother positively charged lipids, and a polyanionic substance. Accordingto the method, premade liposomes are contacted with the polyanionicsubstance in an aqueous environment. The precise nature of the complexformed is determined by the chemical composition of the positivelycharged liposomes used and by the molar ratio of total positive chargeson the liposome, to the total negative charges on the polyanion. Precisetuning of these compositional aspects determines the biological activityof the final product produced. The advantages of this methodology overother liposome technology commonly known in the art are that the newmethod results in up to 100% entrapment of the biologically activesubstance, the entrapped material does not leak out in storage, and thecomplex has unique biological properties not shared by liposomeencapsulated material prepared in the conventional manner. Furthermore,by utilizing double-coated complexes, preferential delivery to aspecific site in the body can be obtained in vivo, to ultimately providesite-specific intracellular delivery via the positively-charged lipidcomplex portion of the double-coated complex.

A. Definitions

An aliphatic chain comprises the classes of alkyl, alkenyl and alkynyldefined below. A straight aliphatic chain is limited to unbranchedcarbon chain radicals.

Alkyl refers to a fully saturated branched or unbranched carbon chainradical having the number of carbon atoms specified, or up to 22 carbonatoms if no specification is made. For example, alkyl of 1 to 8 carbonatoms refers to radicals such as methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, and octyl, and those radicals which are positionalisomers of these radicals. Lower alkyl refers to alkyl of 1 to 4 carbonatoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,secbutyl, and tert-butyl. Alkyl of 6 to 24 carbon atoms includes hexyl,heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl,heneicosyl, docosyl, tricosyl and tetracosyl.

Alkenyl refers to any branched or unbranched unsaturated carbon chainradical having the number of carbon atoms specified, or up to 22 carbonatoms if no limitation on the number of carbon atoms is specified; andhaving 1 or more double bonds in the radical. Alkenyl of 6 to 24 carbonatoms is exemplified by hexenyl, heptenyl, octenyl, nonenyl, decenyl,undecenyl, dodenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl,heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosoenyl,docosenyl, tricosenyl and tetracosenyl, in their various isomeric forms,where the unsaturated bond(s) can be located anywhere in the radical.

Alkynyl refers to hydrocarbon radicals of the scope of alkenyl, buthaving 1 or more triple bonds in the radical.

An antigen is any substance to which an organism can elicit an immuneresponse.

Antisense refers to a nucleotide sequence that is complementary to aspecific sequence of nucleotides in DNA or RNA.

Aryl refers to phenyl or naphthyl.

Aralkyl of 7 to 11 carbon atoms refers to a radical having an alkylgroup to which is attached a benzene ring such as the benzyl radical,phenethyl, 3-phenylpropyl, or the like.

Biologically active substance refers to any molecule or mixture orcomplex of molecules that exerts a biological effect in vitro and/or invivo, including pharmaceuticals, drugs, proteins, vitamins, steroids,polyanions, nucleosides, nucleotides, polynucleotides, etc.

Buffers referred to in this disclosure include “Tris,” “Hepes”, and“PBS.” “Tris” is tris(hydroxymethyl)aminomethane, and for the purposesof the preferred embodiments of this invention is used at about pH 7.“Hepes” is N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, alsoused here as a buffer at about pH 7. Phosphate-buffered saline, or“PBS,” is 10 mM sodium phosphate and 0.9 wt. % NaCl, used as an isotonicphysiological buffer at pH 7.4.

A cell is any one of the minute protoplasmic masses which make uporganized tissue, comprising a mass of protoplasm surrounded by amembrane including nucleated and unnucleated cells and organelles. Anintact cell is a cell with an intact membrane that has not released itsnormal intracellular components such as enzymes, organelles, or geneticmaterial. A viable cell is a living cell capable of carrying out itsnormal metabolic functions.

A complex (or a liposome complex) is defined as the product made bymixing pre-formed liposomes comprising a compound of Formula I with apolyanion (e.g., polynucleotide) or some other macromolecule containingmultiple negative charges. Such a complex is characterized by aninteraction between the polyanion and lipid components that results inthe elution of the polyanion and liposome together as substantially oneentity through a gel filtration column that separates on the basis ofthe Stokes' radius or by some other separation procedure.

A charge ratio refers to a quantitative relationship between the netpositive charges contributed by the lipid and the net negative chargescontributed by the polyanion in a complex. The charge ratio herein isexpressed as positive to negative, i.e., 5:1 means five net positivecharges on the lipid per net negative charge on the polyanion.

Double-coated complexes are prepared from liposome complexes bearing anet positive charge. Liposome complexes bearing a net positive chargeare prepared as described in the preceding paragraph, using a greatermolar amount of positively charged lipid than the molar amount ofnegative charge contributed by the polyanion. These positively chargedcomplexes are mixed with negatively charged lipids to produce thedouble-coated complexes. If sufficient negatively-charged lipid isadded, the final complex has a net negative charge. This definitionincludes liposomes that have further modifications on the surface, suchas the incorporation of antibodies or antigens therein.

DOTMA is the most preferred lipid of Formula I, known asN-(2,3-di(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride. DOTMA vesicles are liposomes made from DOTMA.

DNA represents deoxyribonucleic acid, which may optionally compriseunnatural nucleotides. DNA may be single stranded or double stranded.

Drug refers to any therapeutic or prophylactic agent other than a foodwhich is used in the prevention, diagnosis, alleviation, treatment, orcure of disease in man or animal. (Therapeutically usefulpolynucleotides and polypeptides are within the scope of this definitionfor drugs).

Intracellularly means the area within the plasma membrane of a cell,including the cytoplasm and/or nucleus.

A lipid of Formula I is to be understood as the class of lipids setforth in the Summary of the Invention. Exemplary cyclic structuresrepresented by two or three of R³, R⁴ and R⁵ are quinuclidino,piperidino, pyrrolidino and morpholino.

A liposome formulation is a composition of matter including a liposome,which includes a material encapsulated in the liposome, for diagnostic,biological or therapeutic use.

A liposome-polyanion complex is a composition of matter produced bycontacting a solution of polyanion with a preparation of cationicliposomes produced from a compound of Formula I (with optional co-lipidsas appropriate).

Optional or optionally means that the subsequently described event orcircumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not.

An optional co-lipid is to be understood as a structure capable ofproducing a stable liposome, alone, or in combination with other lipidcomponents, and is preferably neutral, although it can alternatively bepositively or negatively charged. Examples of optional co-lipids arephospholipid-related materials, such as lecithin,phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin,cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate,dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine(DOPE), palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE) anddioleoylphosphatidylethanolamine4-(N-maleimido-methyl)cyclohexane-1-carboxylate (DOPE-mal). Additionalnon-phosphorous containing lipids are, e.g., stearylamine, dodecylarine,hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecylstereate, isopropyl myristate, amphoteric acrylic polymers,triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylatedfatty acid amides, dioctadecyldimethyl ammonium bromide and the like.

A pharmaceutical formulation is a composition of matter including adrug, for therapeutic administration to a human or animal.

A pharmaceutically acceptable anion is an anion which itself isnon-toxic or otherwise pharmaceutically acceptable and which does notrender the compound pharmaceutically unacceptable. Examples of suchanions are the halide anions, chloride, bromide, and iodide. Inorganicanions such as sulfate, phosphate, and nitrate may also be used. Organicanions may be derived from simple organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid,malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid,citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethane sulfonic acid, p-toluenesulfonic acid, and thelike.

A polyanion is a biologically active polymeric structure such as apolypeptide or a polynucleotide, wherein more than one unit of thepolymer bears a negative charge and the net charge of the polymer isnegative.

A polynucleotide is DNA or RNA containing more than one nucleotide.Polynucleotides are intended to include ppp(adenyl 2′→5′)_(n) adenylate,n≧2, represented by 2-5A. A polynucleotide comprising riboinosinic acidand ribocytidylic acid is called poly IC. Polynucleotides are those thatcan be made by chemical synthetic methodology known to one of ordinaryskill in the art, or by the use of recombinant DNA technology, or by acombination of the two.

A polypeptide is a biologically active series of two or more amino acidscoupled with a peptide linkage.

RNA represents ribonucleic acid which may optionally comprise unnaturalnucleotides. RNA may be single stranded or double stranded.

A suitable aqueous medium for forming liposomes from the dried lipidfilm is to be understood as, for example, water, an aqueous buffersolution, or a tissue culture media. For example, a suitable buffer isphosphate buffered saline, i.e., 10 mM potassium phosphate having a pHof 7.4 in 0.9% NaCl solution. The pH of the medium should be in therange of from about 2 to about 12, but preferably about 5 to about 9,and most preferably about 7.

A suitable solvent for preparing a dried lipid film from the desiredlipid components is to be understood as any solvent that can dissolveall of the components and then be conveniently removed by evaporation orlyophilization. Exemplary solvents are chloroform, dichloromethane,diethylether, cyclohexane, cyclopentane, benzene, toluene, methanol, orother aliphatic alcohols such as propanol, isopropanol, butanol,tert-butanol, iso-butanol, pentanol and hexanol. Mixtures of two or moresolvents may be used in the practice of the invention.

A stable transfectant is a living cell into which DNA has beenintroduced and become integrated in the genomic DNA of that cell

Topical administration includes application to any surface of the body,including ocular administration and administration to the surface of anybody cavities.

Transdermal administration is administration through the skin with asystemic effect.

Transfection refers for the purposes of this disclosure to theintroduction of DNA or RNA into a living cell

Unnatural nucleotides include those which are commercially available orwhich can be readily made by means known to those of ordinary skill inthe art.

“Z” refers to the cis form of the aliphatic radicals in Formula I.

The compounds of this invention may be prepared as a racemic mixture ofD,L-isomers or as the individual D or L isomer. Because of theavailability of D or L starting materials, certain of these compoundsare readily prepared as the individual isomer. However, unless thespecific isomer is designated, it should be understood that thisinvention covers both the pure Dor L-isomers as well as theD,L-racemate.

Compounds of Formula I have one asymmetric site, (marked above as *),and thus can exist as a pair of optical isomers. Individual isomers ofcompounds of Formula I are named herein using the IUPAC R-S convention,sometimes called the “sequence rule.” A description of the R-Sconvention may be found, for example, in “Introduction to OrganicChemistry” by A. Streitwieser, Jr. and C. Heathcock, (Macmillan Pub.Co., New York, 1976), pages 110-114. Where appropriate, the opticalactivity of a compound may be indicated by (+) or a (−) for theindividual isomers, or (±) for the racemic mixture, referring to thedirection in which a solution of the compound rotates a plane ofpolarized light. For the purposes of the appended claims, it should beunderstood that racemic mixtures of the compounds of Formula (I) as wellas either isomer taken alone are within the scope of this invention.

B. Utility

The compounds of Formula I are particularly useful in the preparation ofliposomes, but may be used in any of the many uses for which cationiclipids find application. For example, they may be used in industrialapplications, in food or feeds, in pharmaceutical formulations,cosmetological compositions, or other areas where lipids may beemployed. These compounds may also be used in cosmetology, for example,in makeups, lipstick, eyeshadow material, fingernail polishes, bodylotions, moisturizing creams, and the like. They may also be used forapplication to the hair, either alone or in combination with othermaterials, such as in shampoos, hair conditioners, permanent waveformulations or hair straighteners, or as components in hair creams,gels, and the like.

Of particular interest is the use of these compounds in pharmaceuticalformulations, particularly topical formulations such as ointments, gels,pastes, creams, and the like; and more particularly for the preparationof pharmaceutical formulations containing liposomes. The consistency ofthe formulation depends on the amount of aqueous solution used to makethe formulation. In such formulations containing compounds of thisinvention, drugs which are insoluble or only sparingly solublethemselves in aqueous solutions can be solubilized so that a greaterconcentration of drug can be presented to the body.

In pharmaceutical formulations, these compounds may be used in thosecontexts where cationic lipids are acceptable for the formulation ofcreams, pastes, gels, colloidal dispersions, and the like. Foradditional information, reference is made to Remington's PharmaceuticalSciences, 17th Edition, Mack Publishing Company, Easton, Pa. (1985), orany other standard treatise on pharmaceutical formulations.

Other aspects of this invention are directed to the finding thatformulations comprising the compounds of Formula I are useful forachieving desirable intracellular delivery of specific biologicallyactive substances, such as nucleosides, nucleotides, oligoandpoly-nucleotides, steroids, peptides and proteins, and other appropriatenatural or synthetic molecules or macromolecules. The intracellulardelivery can be into the cytoplasm, into the nucleus, or both. Suchintracellular delivery can be achieved in tissue culture and may be usedas an aid in transfecting cells with desired polynucleotide sequences(e.g., deoxyribonucleic acid, DNA) to aid in cloning of specificsequences. Thus, formulations comprising: (1) compounds of Formula I,and (2) DNA or complementary DNA (cDNA)—in appropriate plasmidscontaining promoters, enhancers and the like as desired—, can beutilized to achieve transfection of cells and to obtain stabletransfectants as part of the process of cloning (via recombinant DNAtechnology well known to those familiar in the art) various desiredsequences to yield the corresponding expressed products (e.g., proteinsand peptides). The technology of utilizing a compound of Formula I orother positively-charged lipid formulation to achieve efficienttransfection and to obtain stable transfectants with the desired DNAsequences can enhance the ability to achieve the desired end result ofthe cloning procedure. This technology provides a less toxic and moreefficient route for the delivery of polynucleotides to cells than otherpresently-used techniques such as calcium phosphate precipitation.

Intracellular delivery can also be achieved in the whole organism andmay be useful in several diverse applications. For example,enzyme-replacement therapy can be effected by direct intracellularintroduction of the desired enzymes, or by appropriate transfection ofcells with a DNA sequence encoding the desired protein, with theappropriate promoters and the like included so as to give sufficientgene expression. If desired, inducible promoters can be employed toallow control in turning on or turning off the gene of interest. Otherapplications of intracellular delivery that can be achieved employingthe compounds of Formula I or other positively-charged lipidformulations for transfection of DNA include but are not limited tohormone replacement therapy (e.g., insulin, growth hormone, etc.), bloodcoagulation factor replacement therapy, replacement therapy for otherblood disorders such as β-thalassemia or other hemoglobin deficiencies,adenosine deaminase deficiency, neurotransmitter replacement therapy,and the like. Another application utilizing such formulations to enhanceintracellular delivery includes the delivery of “antisense” RNAoligomers to selectively turn off expression of certain proteins.Compounds of this invention can also be used to deliver biologicallyactive materials across the blood brain barrier.

Formulations comprising the compounds of Formula I can also be used totransfect and transform cells in vitro to introduce a desired traitbefore implantation of the transformed cells into the whole organism. Anexample of this application is to transfect bone marrow cells with adesired gene, such as one coding for normal adult hemoglobin sequencesto correct the deficiency in patients with disorders such asβ-thalassemia, adenosine deaminase deficiency, and sickle-cell anemia.The bone marrow cells can be transfected in vitro, and then theappropriately transfected cells can be transfused into the marrow of thepatient. Alternatively, the cells can be transfected in vivo asdescribed herein. Procedures such as calcium phosphate precipitation aremuch less efficient in effecting such transfections, making themunsuitable for practical use. Other means of achieving transfection thathave been applied in vitro include the use of viral vectors (such asSV-40 and retroviruses). However, these viruses are oncogenic and thuscannot be safely used for transfecting cells in vivo or in vitro forultimate transfusion in vivo.

Intracellular delivery utilizing formulations of compounds of Formula Iis also useful for delivery of antiviral compounds (such as proteaseinhibitors, nucleoside derivatives, nucleotides, or polynucleotides suchas 2-5A); anticancer compounds (including but not limited tonucleosides/nucleotides such as 5-fluorouracil, adenosine analogs,cytosine analogs, and purine analogs); antibiotics such asanthracyclines (for example adriamycin and daunomycin) and bleamycin;protein antibiotics such as neocarzinostatin, marcomomycin, andauromomycin; alkylating agents such as chlorambucil, cyclophosphamide,nitrosoureas, melphalan, aziridines, alkyl alkanesulfonates; platinumcoordination compounds; folate analogs such as methotrexate; radiationsensitizers; alkaloids such as vincristine and vinblastine;cytoskeleton-disrupting agents; differentiating agents; and otheranticancer agents. This aspect of the invention can be particularlyuseful in overcoming drug resistance such as caused by reduced uptakemechanisms of the drug by the cells.

Further selectivity can be achieved by incorporating specific moleculessuch as antibodies, lectins, peptides or proteins, carbohydrates,glycoproteins, and the like, on the surface of the liposome vesicles,which can then serve to “target” the drugs formulated with the compoundsof Formula I to desired tissues bearing appropriate receptors or bindingsites for the ligand attached to the vesicle surface. Furtherselectivity can also be achieved by coating the liposome vesicles with aneutral or negatively-charged optional co-lipid (to eliminatenon-specific adsorption to cells) before addition of the targetingligand as described above.

The use of formulations comprising compounds of Formula I or otherpositively-charged lipid formulations of polynucleotides (including DNAand RNA) for intracellular delivery is superior than other availablemethodology, such as calcium phosphate coprecipitation, or polylysine orDEAE-dextran complexation of polynucleotides, as the formulations ofthis invention are much less toxic and deleterious to the living cellsthan are the other above mentioned procedures. Furthermore, theformulations using compounds of Formula I are much more efficient intransfecting cells. Additionally, the use of liposomes made from thecompounds of Formula I to effect intracellular delivery of the liposomecontents is superior to the use of polyethyleneglycol (PEG) orglycerol-induced fusion of ordinary neutral or negatively-chargedvesicles to cells, because the vesicles of the compounds of Formula I donot require the use of the PEG or glycerol as fusion-inducing agents.These agents are highly deleterious to the viability and integrity ofcells.

Another method that has been employed to induce fusion of liposomes withcells involves incorporation of viral fusion proteins (such as thefusion protein from Sendai virus) on the liposome surface. However, suchtechniques are not only tedious but they also can result in formation ofantibodies by the animal against the viral proteins, thus severelylimiting the utility of this approach.

Other applications of the formulations of this invention comprising thecompounds of Formula I relate to localized delivery of drugs through thestratum corneum, and to transdermal delivery of drugs. Liposome vesiclescomprising the compounds of Formula I can serve to introduce certaincompounds into and through the stratum corneum. Depending on the degreeof penetration enhancement (which is also influenced by the drug and theincorporation of other components in the liposome, such as phospholipidbilayer perturbing agents such as phosphatidylethanolamine, Azone®, andlysolecithin), the formulations can serve to enhance a localized effectof the drug. This enhancement would be applicable to the treatment of alocalized outbreak of herpes simplex virus type 1 or 2 with aninterferon or an interferon inducer, and/or with a nucleoside such as anacyclic guanosine nucleoside analog such as acyclovir or9-(1,3-dihydroxy-2-propoxymethyl)guanine, or9-(1,3-dihydroxy-2-propoxymethyl) guanine dipalmitate. In other cases,the liposomes comprising compounds of Formula I can serve to enhancesystemic uptake of the drug by transdermal absorption, for example aswith topical applications of Synalar® in DOTMA formulations.

Another application of certain formulations comprising the compounds ofFormula I is the enhancement of a specific immune response, such ashumoral and/or cellular immunity, to an antigen of interest which isincorporated in the lipid-containing vesicles. Thus, such preparationscan serve as specific adjuvants for vaccines (including viral,bacterial, rickettsial, parasitic, and cancer vaccines), antigenpreparations, as well as other proteins or peptides including syntheticpeptides of interest. Additional components may be included to furtherenhance the immune response, e.g., immunostimulants such as muramyldipeptide/analogs. N-acetylmuramyl-L-threonyl-D-isoglutamine may beparticularly useful here.

C. Dosage and Administration

Administration of the active compounds and salts described herein can bevia any of the accepted modes of administration for the biologicallyactive substances that are desired to be administered. These methodsinclude oral, topical, parenteral, ocular, transdermal, nasal, and othersystemic or aerosol forms.

Depending on the intended mode of administration, the compositions usedmay be in the form of solid, semi-solid or liquid dosage forms, such as,for example, tablets, suppositories, pills, capsules, powders, liquids,suspensions, or the like, preferably in unit dosage forms suitable forsingle administration of precise dosages. The compositions will includea conventional pharmaceutical carrier or excipient and an activecompound of Formula I or the pharmaceutically acceptable salts thereofand, in addition, may include other medicinal agents, pharmaceuticalagents, carriers, adjuvants, etc.

Topical formulations composed of compounds of Formula I, other lipidmaterial, other penetration enhancers, phosphatidylethanolamine andbiologically active drugs or medicaments can be applied in many ways.The solution can be applied dropwise, from a suitable delivery device,to the appropriate area of skin or diseased skin or mucous membranes andrubbed in by hand or simply allowed to air dry. A suitable gelling agentcan be added to the solution and the preparation can be applied to theappropriate area and rubbed in. Alternatively, the solution formulationcan be placed into a spray device and be delivered as a spray. This typeof drug delivery device is particularly well suited for application tolarge areas of skin, to highly sensitive skin or to the nasal or oralcavities.

For oral administration, a pharmaceutically acceptable non-toxiccomposition is formed by the incorporation of any of the normallyemployed excipients, such as, for example pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium, carbonate, and the like. Suchcompositions take the form of solutions, suspensions, tablets, pills,capsules, powders, sustained release formulations and the like. Theexact composition of these formulations may vary widely depending on theparticular properties of the drug in question. However, they willgenerally comprise from 0.01% to 95%, and preferably from 0.05% to 10%,active ingredient for highly potent drugs, and from 40-85% formoderately active drugs.

Parenteral administration is generally characterized by injection,either subcutaneously, intramuscularly or intravenously. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like. Inaddition, if desired, the pharmaceutical compositions to be administeredmay also contain minor amounts of non-toxic auxiliary substances such aswetting or emulsifying agents, pH buffering agents and the like, such asfor example, sodium acetate, sorbitan monolaurate, triethanolamineoleate, etc.

The amount of active compound administered will of course, be dependenton the subject being treated, the type and severity of the affliction,the manner of administration and the judgment of the prescribingphysician. In addition, if the dosage form is intended to give asustained release effect, the total dose given will be integrated overthe total time period of the sustained release device in order tocompute the appropriate dose required. Although effective dosage rangesfor specific biologically active substances of interest are dependentupon a variety of factors, and are generally known to one of ordinaryskill in the art, some dosage guidelines can be generally defined. Formost forms of administration, the lipid component will be suspended inan aqueous solution and generally not exceed 30% (w/v) of the totalformulation. The drug component of the formulation will most likely beless than 20% (w/v) of the formulation and generally greater than 0.01%(w/v).

In general, topical formulations using a compound of Formula I areprepared in gels, creams or solutions having an active ingredient in therange of from 0.001% to 10% (w/w), preferably 0.01 to 5%, and mostpreferably about 1% to about 5%. (Of course, these ranges are subject tovariation depending upon the potency of the drug, and could inappropriate circumstances fall within a range as broad as from 0.0001%to 20%.) These guidelines would pertain, for example, to topicallyapplied transglutaminase inhibitors such as5-(N-benzyloxycarbonyl-L-paratyrosinamidomethyl)-3-chloro-4,5-dihydroisoxazole,also named in its preferred form as(S,S)-2-1(1-benzyloxymethanamido)-N-[(3-chloro-4,5-dihydroisoxazol-5-yl)methyl]-3-(4-hydroxyphenyl)-propanamide,which could be applied twice daily in a formulation containing 2.5%active ingredient. Similarly, for Butoconazole nitrate (see Example 4,Part 4), the preferred amount of active ingredient will be about 1%.Another example of a topical formulation is the class of 5-lipoxygenaseantipsoriatic agents, such as lonapalene, which is preferably formulatedwith about 1% active ingredient. In all of these exemplary formulations,as will other topical formulations, the total dose given will dependupon the size of the affected area of the skin and the number of dosesper day. The formulations can be applied as often as necessary, butpreferably not more than about 3 times per day.

Other topical formulations including a compound of Formula I also fallwithin these guidelines. The acyclic guanosine nucleoside analog,acyclovir, or 9-(1,3-dihydroxy-2-propoxy methyl)guanine dipalmitate(also known as DHPG dipalmitate), is topically applied in formulationsusing most preferably about 3% (w/w) active ingredient. Likewise, atopical formulation of ketorolac, i.e.,5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2-a]pyrrole-1-carboxylic acid, can beused having about 3% active ingredient; or about 0.5% active ingredient,as a salt, for ocular administration. Interferon (α-, β orγ-interferons, or a mixture of any of these), is used at 100 to 10⁹units per gram of cream, gel, lotion, ointment or liposome formulationcontaining a compound of Formula I, more preferably from 10⁴ to 10⁸units per gram, and most preferably from 10⁵ to 10⁷ units per gram. Theacyclic nucleoside analog may be used alone or in combination with anyor all of the interferons. The gel, cream, ointment, lotion, or liposomeformulation containing the nucleoside and/or interferon is appliedtopically or intravaginally to the area of the viral outbreak, and canbe applied 1 to 6 times daily, preferably 1 to 4 times daily, for asmany days as needed, typically from 2 to 8 days.

The oral DHPG dipalmitate, or an analog thereof, can be formulated usinga compound of Formula I, as a solution, tablet or capsule, and beadministered as a dose of about 500 mg active ingredient per day per 70kg person. Doses can be given up to several times daily, but are moretypically given 3 to 5 times over a period of a week.

The systemic DHPG dipalmitate, or an analog thereof, can be formulated,using a compound of Formula I, as a solution and administered at a doseof about 350 mg per day per 70 kg person. Doses can be given daily, butare more generally given 3 to 5 times per week and after the first weekthe patient's condition is evaluated.

For the inotropic agent,N-cyclohexyl-N-methyl-4-(2-oxo-1,2,3,5-tetrahydroimidazo-[2,1-b]quinazolin-7-yl)-oxybutyramide,formulated with a compound of Formula I, an appropriate dosage range fororal or intravenous administration would be from about 0.1 to about 25mg/kg, and preferably from about 1 to about 10 mg/kg.

Prostaglandin analog formulations using a compound of the Formula I aregenerally administered in the range of about 1 mg of active ingredientper 70 kg person.

For suitable nicardipine dosage and administration, the full disclosureof U.S. Ser. No. 06/877,812, filed Jun. 24, 1986, is hereby incorporatedby reference.

Regarding vaccine administration, to achieve the desired immuneresponse, the antigen in the formulation comprising a compound ofFormula I is administered to an animal or mammal in need thereof, eitherby injection (such as subcutaneous, intraperitoneal, intramuscular orintravenous) or orally, or by intranasal administration. The formulationof a vaccine using the compounds of Formula I described herein willemploy an effective amount of antigenic material. That is, there will beincluded an amount of antigen which, in combination with the adjuvants,will cause the subject to produce a specific and sufficientimmunological response so as to impart protection to the subject fromthe subsequent exposure to the material or organism against which thevaccine is intended to be effective. Alternatively, the antibody willcombine with a hormone or naturally occurring material in such a way asto alter biological processes such as growth.

No single dose designation can be assigned which will provide specificguidance for each and every antigen which may be employed in thisinvention. The effective amount of antigen will obviously be a productof its inherent activity and molecular weight, and will be a function ofthe degree to which the specific antigen can be purified from itssource. It is contemplated that the lipid or liposome formulations ofthis invention may be used in conjunction with whole cell or virusvaccines as well as purified antigens or subunit or peptide vaccinesprepared by recombinant DNA techniques or synthesis.

However, as a general matter, the amount of antigen used can range from0.01 μg/kg to 1 mg/kg, and more preferably from 0.1 to 200 μg/kg. Aprimary vaccination is administered, and if desired this can be followedby one or more booster vaccinations given usually from 2 weeks toseveral months after the primary vaccination. If desired, boostervaccinations can be administered at regular intervals (such as on ayearly schedule or a schedule of every two to three years.). Theantigens of herpes viruses, influenza viruses, malaria parasites,hepatitis viruses (such as hepatitis B surface or pre-S antigens), orretroviruses such as human immunodeficiency viruses, may be particularlyuseful for preparing vaccines of this invention. IfN-acetylmuramyl-L-threonyl-D-isoglutamine is included to further enhancethe response, the dose of this component can range from 0.001 to 1mg/kg, and more preferably from 0.01 to 0.5 mg/kg.

D. Specifically Preferred Embodiments

1. Lipids, Liposomes, and Pharmaceutical Formulations

The preferred compounds of Formula I are those wherein R¹ and R² areapproximately the same length and are independently alkyl or alkenyl of10 to 20 carbon atoms; R³, R⁴, and R⁵ are methyl or ethyl; n is 1 to 4;and X is a halide ion. In the more preferred group, n is 1, and R¹ andR² are the same length.

The following racemic compounds and the optical isomers thereof areexamples of preferred compounds:

N-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-tri-methylammoniumchloride (DOTMA, the most preferred compound);

N-(2,3-di-octadecyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride;

N-(2,3-di-(4-(Z)-decenyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride;

N-(2,3-di-hexadecyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride(“BISHOP”);

N-(2,3-di-decyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride;

N-(2-hexadecyloxy-3-decyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride;

N-(2-hexadecyloxy-3-decyloxy)-prop-1-yl-N,N-dimethylamine hydrochloride;

N-(9,10-di-decyloxy)-dec-1-yl-N,N,N-trimethylammonium chloride;

N-(5,6-di-(9-(Z)-octadecenyloxy))-hex-1-yl-N,N,N-trimethylammoniumchloride; and

N-(3,4-di-(9-(Z)-octadecenyloxy))-but-1-yl-N,N,N-trimethylammoniumchloride.

Certain aspects of this invention relate to the use of liposomes madefrom the most preferred compound of Formula I, which liposomes arereferred to herein as “DOTMA vesicles.” DOTMA vesicles can be made withpure DOTMA, or with DOTMA in combination with other compounds of FormulaI or other classes of positively charged lipids, for example similar tothose of Formula I but containing ester instead of ether linkages on R¹and/or R². Also, optional co-lipids can be combined with compounds ofFormula I, such as DOPC and DOPE and the like. Optional co-lipids, suchas DOPC and DOPE, can be mixed with the DOTMA analog in quantities equalto from 0 to 99%, more preferably from 10 to 90% and most preferablyfrom 30 to 70%.

A list of optional co-lipids which can be used includes, for example,ternary or complex lipids, glycerides, cerides, etholides and sterides;i.e., any of several compounds having a hydrophilic and a lipophilicgroup, wherein the hydrophilic group is a phosphate, carboxylate,sulfate, amino, hydroxyl or choline group; and the lipophilic group isan alkyl or alkenyl, polyoxyalkylene or aromaticor cycloalkylsubstitutedalkyl group. Polyethyleneoxy or glycol groups may be used. Additionallipids suitable for incorporation into these formulations can be foundin the McCutcheon's Detergents and Emulsifiers and McCutcheon'sFunctional Materials, Allured Pub. Co., Ridgewood, N.J., U.S.A.

Preferred optional co-lipids are phospholipidrelated materials such as,for example, lecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides,dicetyl phosphate, phosphatidylcholine anddipalmitoylphosphatidylcholine. Additional, non-phosphorus-containinglipids are, for instance, cetyl palmitate, glyceryl ricinoleate,hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers,triethanolamine-lauryl sulfate, alkanoyl-aryl sulfonates, and the like.

Additional additives may be long chain alcohols and diols; sterols, forexample, cholesterol; phosphoric esters of fatty alcohols, for example,sodium dicetyl phosphate; alkylsulfates, for example, sodium cetylsulfate; certain polymers such as polypeptides; positively-chargedlipids such as stearylamine or dioctadecyldimethyl ammonium bromide; andproteins

Typically, the aqueous liposome formulations of this invention willcomprise 0.01 to 10% drug by weight (i.e., 10% is 100 mg drug per ml), 1to 20% lipid by weight comprising a compound of Formula I in a quantityof 1 to 100% of this lipid component by weight, and an aqueous solution,that is, water which may or may not contain salts and buffers, in aquantity sufficient to make 100% by volume. Particularly preferred areformulations which comprise 0.1 to 5% drug and a lipid componentcomprising a compound of Formula I in a quantity of 50% or more byweight of the lipid component. Most preferred is a formulationcomprising 1 to 5% drug by weight; up to 20% by weight of a lipidcomponent, in turn comprising 10 to 100% by weight of a compound ofFormula I; and an amount of aqueous solution sufficient (q.s.) to make100% by volume.

Formulations of this invention, particularly liposomes, made with thecompounds of Formula I will exhibit the properties of a positivelycharged entity when compounds of Formula I, comprising 1% or more byweight of the total weight, are used with a neutral liposome-formingmaterial. Thus, other excipients, optional co-lipids and the like whichare used for making liposomes, can be used in these formulations. Onemay use any combination of optional co-lipids with the compounds ofFormula I so long as there is 1% or more of a compound of Formula Ipresent in the formulation.

2. Liposome-Polyanion Complexes

Although the charge ratio is an important factor to consider in thepreparation of DNA/DOTMA or RNA/DOTMA liposome complexes, it can varyconsiderably, depending upon the application. The charge ratio must begreater than 1:1, but is preferably in the range of 1000:1 to 1:1; morepreferably 20:1 to 1:1; and most preferably 5:1 to 2:1.

The most appropriate ratio can routinely be determined by one ofordinary skill in the art. The precise ratio necessary for the bestresults must, however be optimized for each individual example. Morespecifically, the number of moles of anion in a stock solution of anionand the number of moles of cation in the stock liposome solution mustfirst be quantitatively determined. When the ratio of cation to anionranges between 0.5:1 and 2:1, significant turbidity generally developsupon mixing. (Significant turbidity is a net turbidity greater than thecombined optical density of either component taken alone. The opticaldensity is usually measured at 400 nm, but this wavelength is notcritical.) This turbidity is a direct indication of complex formationbetween the cationic liposomes and the polyanionic substance. When thecharge ratio of the added components is higher than 2 or lower than 0.5,complex formation still occurs, but it is not as readily apparent fromgross changes in light scattering.

Having determined by light scattering that complex formation occursbetween the polyanion and the positively charged liposome, it is thennecessary to determine the ratio of the two components that yields theoptimum biological effect. This is a simple, routine determination toone of ordinary skill in the art. The appropriate starting point wouldbe at the ratio of greatest turbidity. For example, with respect to DNAtransfection, it is necessary to determine the ratio of the two addedcomponents which leads to optimum expression of the transfected DNA.Specifically, with respect to DNA transfection of mouse L cells with apSV2CAT plasmid, it is determined that while optimum light scatteringoccurrs at a ratio of about 1 to 1, the optimum ratio for transfectionoccurrs at a charge ratio of about 2.5:1.

Polynucleotide/lipid complexes prepared as described are extremelyconvenient and useful for the delivery and expression of DNA and RNA intissue culture cells. Similar complexes can be formed withnon-polynucleotide materials for intracellular delivery, if theyintrinsically contain or are otherwise provided with a negative charge.(E.g., ionic molecules can frequently acquire a negative charge byraising the pH; and a negative charge can also be added to a molecule bythe covalent attachment of polyvalent negatively charged material suchas a polynucleotide, or sulfated polydextran.)

3. Double coated complexes

The complexes described hereinabove involve the addition of an excess ofpositively charged lipid material to a polyanion. The complex soproduced bears a net positive charge enabling it to spontaneouslyintereact with negatively charged surfaces, such as the surface oftissue culture cells. Since most biologically interesting surfaces arenegatively charged, this general approach is broadly applicable forinducing negatively charged materials to intereact with such negativelycharged biological surfaces.

According to another aspect of the invention, it has been found usefulto prepare complexes which do not interact spontaneously with the firstnegatively charged surface with which they come in contact. In order toachieve this result, “double coated” complexes are prepared bycontacting the (positively charged) polyanion-lipid complexes with asecond population of negatively charged liposomes. The quantity ofnegatively charged lipid added in this population exceeds the quantityof positive charged contribued by the initial population of lipid, sothat the net charge on the resulting complexes is negative. Theresulting double-coated complexes do not spontaneously intereact withtissue culture cells.

This methodology has several advantages over conventional liposomeentrapment methods: 1) a relatively high capturing efficiency (up to100% can be obtained by this procedure, compared with other proceduresfor encapsulation of large polyanions that can achieve as little as itentrapment or less; 2) it is simple to manufacture the double-coatedcomplexes from premade stable stocks; and 3) prior complexation withpositive lipids gives unique physical properties to the initialpolyanion of interest that can favorably effect its biological activity.

In addition, this procedure affords convenient preparation of complexesthat associate with cells in a receptor-specific manner (See Example10). This is accomplished by including a coupling reagent in thenegatively charge liposome population. Antibodies, lectins, ligands andthe like can be attached to the double-coated complexes in order totarget them to a specific receptor.

With respect to the preparation of double coated complexes, somenegatively charged liposomes are first prepared. These vesicles mustcontain at least one negatively charged lipid component, such as DOPG,in amounts equal from 1 to 100 mole %, preferably 10 to 90%, and mostpreferably 30 to 70%. A suitable lipid coupling agent, such asmaleimide-PE, can be included in this formulation, depending on whetheran additional covalently coupled surface component is desired in thefinal formulation. Maleimide-PE can be added at a mole percent from 0.01to 20%, preferably 0.1 to 5%. A procedure for producing this type ofcovalent modification is given in the Examples. After preparation of theDNA/DOTMA liposome complexes according to the previous subsection, analiquot of the negatively charged vesicles is added to produced thedouble coated vesicles. The molar ratio of DOTMA to the negativelycharged lipid in the DNA/DOTMA liposome complex can vary considerablydepending upon the desired charge of the final double-coated complex.

4. Transfection of cells with liposome complexes

DNA-liposome complexes can be mixed with cells, either attached to asolid support or free floating in a suspension, under various conditionsto achieve efficient transfection of the DNA. Cells can be grown on asolid support to a density of 100 to 1,000,000 cells per cm², butpreferably 1000 to 100,000 cells per cm², most preferably about 30,000cells per cm², using any conventional growth media or other conditionsrequired to maintain viable cells. Cells in suspension culture can beused at a concentration of from about 100 to about 5×10⁷ cells per ml,preferably from about 10⁵ to about 10⁷ cells per ml, and most preferablyabout 10⁶ cells per ml. The buffer to which the cells are exposed duringthe addition of DNA-liposome complex can vary, however, this buffer mustbe nearly isotonic. The tonicifier can be salts or sugars or otheragents that are compatible with cellular viability in tissue culture.The buffer can contain any other components necessary for viability ofthe cells, but preferably the buffer is free of interfering serum orother protein components during an initial incubation phase which canlast from about 5 minutes to 48 hours, but preferably 1 to 24 hours. Theincubation can proceed at various temperatures including 0 to 55°, butpreferably 15 to 42° and most preferably 20 to 38°. After this initialincubation with DNA-liposomes complex the cells can either be washed ornot and the other components necessary for long term viability can beadded back. The cells can be allowed to grow for an additional period oftime if desired. The amount of DNA added to the cells in the form of aDNA-liposome complex can vary from between 0.001 and 10000 μg per1,000,000 cells, but is preferably in the range of 0.01 to 1000μg/1,000,000 cells, and most preferably 0.1 to 100 μg/1,000,000 cells.

This DNA transfection method can be used as part of a therapeuticprotocol to treat genetic disorders. The treatment can be performed byeither withdrawing cells from the affected patient, transfecting invitro with the appropriate gene and reinjecting the successfullytransfected cells; or by systemically administering the appropriate DNAdirectly into the affected patient with a suitable vehicle that willallow the transfection event to occur in vivo. The in vitro protocol isperformed in the following way adapted from the literature (Anderson WF, Science 226, 401-409 (1984); Williams D A, Orkin S H, Mulligan R C,Proc. Nat. Acad. Sci. 83, 2655-2570 (1986)). A suitable quantity oftissue cells (from 10 million to 10 billion) is extracted from thepatient. The tissue cells can be derived from various organs such asliver, spleen, blood or skin but most probably form bone marrow. Thecells are prepared for tissue culture by trypsinization of the tissue orother means if necessary, grown in an appropriate media for a suitablelength of time (e.g., 1 day to 2 weeks) and then transfected by addingthe DNA/DOTMA liposome complex that is appropriate for the particulargenetic disorder treated and with a composition consistent with themethod described in the preceding paragraphs. The cells are incubatedfor a suitable length of time, approximately 4 to 72 hours, and thesuccessfully transfected cells are washed and reinjected back into theaffected individual. The affected individual can be treated with a highdose of radiation or by some chemical means prior to injection of thetransfected bone marrow cells, in order to kill resident bone marrowcells. This gives the newly transfected cells a selective advantage asthey repopulate the bone marrow. However, this last step may not benecessary, particularly if a selective advantage is built into thetransfected polynucleotide.

The in vivo transfection protocol can be done in the following manner,adapted from Nicolau et al., Proc. Nat. Acad. Sci. 80, 1068-1072 (1983).DNA/DOTMA liposome complexes, or double coated DNA complexes, orcovalently modified double coated complexes are prepared as previouslydescribed. The covalently modified complexes may contain attachedantibodies, proteins, hormones, carbohydrates or other chemicalmodifications so as to target them to the particular cells of interest.For instance, the complexes can contain an antibody to endothelial cellsin order to target the complexes to the endothelial cells; or they cancontain antibody to a particular subpopulation of bone marrow cells inorder to target the complexes to those cells. The administration to theaffected individual can be intravenous (IV), subcutaneous (SC),intraperitoneal (IP), intramuscular (IS), topical, or by aerosol to thenose or lung. The therapeutic protocol can involve either a singletreatment or the complex can be given as often as required. The IV dosecan be given as a bolus or by slow infusion.

IV. REACTION SCHEMES AND PREPARATION METHODS

Except where indicated, the substituents in each reaction scheme arecommensurate in scope with the broadest claim.

This reaction scheme is applicable to the synthesis of the compound ofFormula (I) in racemic or either optically active form. It is applicablewhen n is 1; R³, R⁴ and R⁵ are the same or different and R⁵ is not aryl;where R¹ and R² are the same; and where X_(a) ⁻ and X_(b) ⁻ aredifferent anions.

The p-toluenesulfonate ester of formula (1) is commercially available inracemic and both optically active forms. In some cases, the3-(dialkylamino)-1,2-propane diols of formula (3) are commerciallyavailable in racemic form.

To effect the formation of compounds of formula (2), thep-toluenesulfonate ester of formula (1) is allowed to react with anexcess of an appropriate secondary amine in the absence of a solvent.This mixture is heated between about 25° and about 150° C., morepreferably 75° C. for approximately 1 to about 5 days, preferably about2 days. The amine is added in an excess molar amount, preferably about 2to about 10 times the amount of the p-toluenesulfonate ester of formula(1) being used, more preferably about 5 times that amount.

The diol of formula (3) is then prepared by removal of theisopropylidene group from the amine of formula (2) in acidic methanol ofpH between about 1 to about 5, preferably about pH 3. This mixture isheated between about 25° and about 65° C., preferably about 65° C., forabout 1 to about 5 hours, preferably about 2 hours.

To effect the formation of the compound of formula (4), the diol isdissolved in an appropriate high boiling solvent such as xylene,mesitylene or the like. To this is added an alkali base, such aspotassium tert-butoxide and an alkylating agent of the desired chainlength and degree of unsaturation. For example the p-toluenesulfonateester of oleyl alcohol can be used to effect the addition of9-Z-octadecenyl groups. This mixture is then heated, preferably betweenabout 100° and about 200° C., more preferably to about 140° C., withstirring for approximately 1 to about 5 hours, preferably about 3 hours.The base is added in an excess molar amount, preferably about 2 to about4 times the amount of diol being used, more preferably about 3 timesthat amount. The alkylating agent is also added in an excess molaramount, preferably about 2 to about 4 times the amount of diol beingused, more preferably about 3 times that amount.

The quaternary ammonium compound of Formula I is then prepared bycondensing an alkyl chloride into a reaction vessel containing thecompound of formula (4), after which the reaction vessel is sealed andheated to between about 50° and about 100° C., preferably about 70° C.,for up to 60 hours. This procedure affords the tetraalkylammoniumchloride product of Formula (I).

Alternatively, one quaternary ammonium compound of Formula I can beconverted to another compound of Formula I by exchange of the anioniccounterion (i.e., X_(b) ⁻ for X_(a) ⁻). The quaternary ammonium sulfateof Formula I is prepared by the alkylation of the compound of formula(4) with a dialkylsulfate in an appropriate solvent at or above roomtemperature. Depending on the reactivity of the dialkylsulfate, thismixture can be heated to 150° C. to obtain the product of Formula I.However, with a reactive dialkyl sulfate, room temperature is preferred.The alkylating agent is added in an excess molar amount, preferablybetween 1 and about 3 times the amount of compound of formula (4), morepreferably 2 times that amount.

The quaternary ammonium sulfate of Formula I obtained in this manner canbe converted to a quaternary ammonium chloride of Formula I by anionexchange. A solution of the sulfate form of a compound of Formula I inan appropriate organic solvent is treated with an excess molar amount ofsodium chloride as a saturated solution in water. The two phases aremixed vigorously and allowed to separate. The organic layer is removedand the tetraalkylammonium chloride product of Formula I is isolated.The sodium chloride is used in an excess molar amount, preferably 1 to10 times the amount of the sulfate compound of Formula I, morepreferably about 5 times that amount.

This two-step procedure can be carried out at atmospheric pressure,avoiding the high pressures which can be generated in the precedingcase.

REACTION SCHEME II

Alternatively, optically active compounds of Formula (I) commensurate inscope with Reaction Scheme I, can be prepared in the (S) absoluteconfiguration by this Reaction Scheme II. Where n is 1, the compoundsmost analogous to glycerol, the compounds of this invention can bederived from D-mannitol. The two central hydroxy groups of mannitol arefirst protected by formation of a ketal, for example by formation of theacetonide of formula (7). The four remaining hydroxy groups are thenconverted to ethers of formula (8) using the appropriate long chainalkylating agent. The compound of formula (8) is hydrolyzed to that offormula (9), which is then chemically split into two units of aldehydeof formula (10) of 3 carbon atoms each, wherein two carbon atoms aresubstituted with a long chain alkyl, alkenyl or alkynyl group. Thealdehyde functionality is then converted to a tertiary amine of formula(11) and then further converted to either the acid addition salt or aquaternary ammonium compound of Formula (I). This process is exemplifiedby Reaction Scheme II:

The D-mannitol-3,4-acetonide of formula (7) is prepared in two stepsfrom commercially available D-Mannitol (formula (5)). To effect thistransformation, D-mannitol is allowed to react with 2,2-dimethoxypropanein actone in the presence of an acid catalyst. This produces theD-mannitol-1,2:3,4:5,6-trisacetonide of formula (6), which is partiallyhydrolyzed in aqueous acetic acid to the D-mannitol-3,4-acetonide offormula (7).

To effect formation of compound (8), the acetonide (formula (7)) isdissolved in an appropriate polar solvent such as dimethylformamide,diethylformamide, or the like. To this is added a strong base, such assodium hydride, at room temperature. This mixture is then heated,preferably between about 30° and about 100° C., more preferably to about50° C., with stirring for approximately 30 to about 90 minutes,preferably about 60 minutes. To this is then added an alkylating agentof the desired chain length exemplified by the toluenesulfonate ester ofoleyl alcohol or by 1-bromohexadecane. Following addition of thealkylating agent, the temperature is increased to between about 50° andabout 150° C., preferably about 90° C., with additional stirring over aperiod of up to 2 hours, preferably about 1 hour. In the first addition,the base is added in an equal molar amount to the amount of acetonidebeing used and the alkylating agent is added in an equal molar amount.This sequence of adding a molar amount of base with heating followed bya molar amount of the alkylating agent with heating and stirring isrepeated four times (for a total of five times) in order to effect theformation of compound (8).

Compound (9) is made by hydrolyzing the ketal, illustrated by theacetonide of formula (8). The hydrolysis is carried out as a singlephase reaction using a polar, water soluble organic solvent, such astetrahydrofuran. Preferably, the hydrolysis will be effected by means ofa 10% solution of water in trifluoroacetic acid. The solution of theacetonide of formula (8) in organic solvent and aqueous acid solutionare stirred for up to 3 hours, preferably one hour at a slightlyelevated temperature, approximately 25°-70° C. preferably about 50° C.The solvent is then evaporated and residual acid removed by azeotropicdistillation using a solvent such as toluene.

The aldehyde (10) is made by treating diol (9) with an oxidant,preferably one such as lead tetraacetate, in a solvent best illustratedby chloroform. A slight molar excess of lead tetraacetate is used toeffect the reaction. The mixture is stirred at about ambient temperaturefor up to 4 hours, preferably about 2 hours, at which time the excesslead tetraacetate is quenched by addition of ethylene glycol followedquickly by the addition of a substantial amount of water. The resultingcrude aldehyde is recovered by conventional means and may be usedwithout further purification directly in the next step.

To effect the formation of the amine of formula (11), the appropriateamine hydrochloride, such as, for example, a commercially availablesecondary amine hydrochloride is dissolved in an alcohol, preferablymethanol, to which solution is added a two-thirds molar amount ofanhydrous sodium acetate. This mixture is stirred for about an hour atambient temperature and the resulting sodium chloride is filtered off.The methanol solution is then added to the crude aldehyde of formula(10) from the preceding paragraph. A second solvent, preferablytetrahydrofuran, is then added to this mixture followed by molecularsieves. To this mixture is then added a reducing agent, preferablysodium cyanoborohydride, in a slight molar excess, and the mixturestirred at a slightly elevated temperature, preferably about 40 to about60° C., for up to 3 days. This product (11) is then converted to theacid addition salt of formula (I) where R⁵ is hydrogen by the additionof the appropriate acid such as hydrochloric acid in an organic solvent.

Alternatively, when R⁵ is not hydrogen, the quaternary ammonium compoundis then prepared by condensing an alkylating agent into a reactionvessel containing the amine material (11), after which the reactionvessel is sealed and heated to between about 50° and about 100° C.,preferably about 70° C., for about 1 to about 5 days, preferably about 2days. This procedure affords the tetraalkylammonium chloride product ofFormula I.

Furthermore, when n is 1 and R¹ and R² are not the same, the compoundsof Formula (I) can be prepared by the flowchart of Reaction Scheme IIIwhich follows.

In this Reaction Scheme, R¹ and R² can be the same or different, and R⁵is not aryl. The 1,3:4,6-di-O-benzylidine-D-mannitol of formula (12) iscommercially available and is converted to the di-O-benzyl ether offormula (13) by the action of between 5 and 15, preferably about 10,molar equivalents of potassium hydroxide in benzylchloride at about 120°to about 160° C., preferably about 140°, for up to 5 hours, preferably 3hours. This, in turn, is then hydrolyzed to the2,5-di-O-benzyl-D-mannitol of formula (14) with aqueous acid in ethanolof pH between 0 and about 4, preferably about 1.5, at reflux for up to10 hours, preferably about 5 hours.

The central hydroxyls of compound (14) were protected as a ketal, suchas the acetonide of compound (15), by the reaction of compound (14) witha ketone under the influence of an acidic copper catalyst. Thus,compound (14) was dissolved in acetone and treated with 0.5 molarequivalents of copper sulfate and a slightly greater amount of sulfuricacid, preferably about 1.2 times that amount.

The two terminal hydroxyls of compound (15) were converted to the ethersof formula (16) by the action of an alkali base such as potassiumtert-butoxide or more preferably potassium hydroxide in an appropriatehigh boiling solvent such as xylene, mesitylene or the like. To this isadded an alkylating agent of the desired chain length and degree ofunsaturation and the mixture is heated between about 100° and about 200°C., more preferably about 140° for up to 20 hours, preferably about 4hours.

The two benzyl groups of compound (16) are then removed by catalytichydrogenolysis in an appropriate solvent such as a mixture oftetrahydrofuran and methanol. A transition metal catalyst such as 10%palladium on carbon is used. The reaction is carried out in anappropriate hydrogenolysis device, in this instance with heating toabout 60° to about 80° C., for about 48 hours under about 60 psi ofhydrogen.

The diol of formula (17) obtained from the preceding hydrogenolysis isetherified in the same manner described above for preparing compound(16).

Once the tetrasubstituted D-mannitol-3,4-ketal of formula (18) isobtained, it is converted to Formula I by the series of steps recitedabove for conversion of formula (8) to the compounds of Formula I.

Those compounds wherein n is 2-8 are prepared from the correspondingtriol. The schematic for this reaction sequence is set forth in ReactionScheme IV which follows. This scheme may also be used for preparingcompounds where n is 1.

In this reaction scheme, R¹ and R² are the same, X is a leaving group,X⁻ is an anion which may optionally correspond to the leaving group X,and n can be 1 to 8.

The compounds of formula 20 are known in the literature or may bepurchased from a chemical supply house or may be prepared by the actionof osmium tetroxide and trimethylamine-N-oxide on the appropriatealkenol of formula (19) in aqueous acetone/tert-butanol at roomtemperature for up to 48 hours, preferably about 20 hours.

The ketal of formula 21, preferably the acetonide, is prepared bydissolving the appropriate triol in acetone with the addition of a smallamount of concentrated sulphuric acid. This reaction may be effected bystirring the solution for up to about 4 hours at room temperature,preferably about 2 hours. The resulting ketal is then recovered by somestandard separatory means.

The unprotected primary hydroxyl group of compound (21) is thenprotected by forming an allyl ether. This reaction is carried out bydissolving the alcohol in a dry dipolar aprotic solvent, such asdimethylformamide. A strong base, such as sodium hydride (an equal molaramount), is added to the alcohol which is stirred at ambient temperaturefor a set period and then warmed to between about 80° and about 100° C.for an equal period. Allyl chloride, in about a 50% molar excess, isthen added at the elevated temperature with stirring. Stirring andheating is continued for another approximately 30 to 120 minutes,preferably about 60 minutes. The product of formula (22) is thenextracted and further purified by chromatographic means.

The ketal of compound (22) is then hydrolyzed by means of a dilutesolution of a strong acid, for example, 1N HCl, the reaction beingcarried out in a polar solvent, such as methanol, ethanol, or the like.Some heat is added to the reaction mixture to effect the hydrolysis.Preferably, a solution is heated to about 50° C. for about 2 hours.

The diol of formula (23) is converted to the diether of formula (24) inthe same manner as described above for conversion of formula (21) toformula (22). Here again the etherfication carried out in a dry dipolaraprotic solvent, such as dimethylformamide, using a strong base, such assodium hydride, and the p-toluenesulfonate ester or halide of theappropriate chain length and degree of unsaturation. The reaction isrepeated twice using a one molar equivalent of alkylating agent eachtime. As described previously, the reaction is effected at an elevatedtemperature, preferably between about 50° and about 150° C., morespecifically at about 90° C.

The allyl ether of formula (24) is then hydrolyzed by means ofWilkinson's catalyst [tris(triphenylphosphine)rhodium chloride] in anacid medium. The solvent should be a polar solvent such as ethanol,preferably with a co-solvent such as tetrahydrofuran. Thetriether/catalyst mixture is refluxed for several hours, preferablyabout 3 hours, at which time additional acid (1N HCl) is added andrefluxing continued for several more hours (approximately 3 to 4). Theseconditions effect hydrolysis of the allyl ether.

The alcohol of formula (25) is then converted to the amine by firstcreating an intermediate p-toluenesulphonate ester of formula (26) towhich is added a dialkylamine to effect formation of the amine compound.By way of illustration, the alcohol is dissolved in a suitable solvent,such as pyridine, to which is added p-toluenesulphonyl chloride. Thismixture is stirred overnight at ambient temperature, then poured intoice water and the product (26) recovered by extractive means. The crudeproduct is immediately dissolved in a dialkylamine, such asdimethylamine, and placed in a sealed container at between about 25° andabout 100° C., preferably about 70° C., for about 1 day to effectformation of the trialkylamine of formula (26).

The trialkylamine is most conveniently converted to an acid additionsalt, preferably a hydrochloride salt, as a means of isolating theproduct.

The quaternary ammonium product Formula I is then prepared in the samemanner as described hereinabove for the conversion of formula (4) toFormula I.

Alternatively, compounds of Formula I can be prepared by the reaction ofcompounds of formula (26) with the appropriate tertiary amine. This isparticularly useful in the synthesis of compounds of Formula I whereinR⁵ is aryl and where the positively charged nitrogen and two or three ofR³, R⁴ and R⁵ are combined to form one or two rings. For example, asolution of (26) and quinuclidine in dichloromethane was sealed in apressure reactor and heated between about 50° and 150° C., preferablyabout 100° C., for up to 5 days, preferably about 2 days. This resultedin the formation of a compound of Formula I containing a bicyclicammonium group.

V. PREPARATIONS AND EXAMPLES Preparation 1(S)-3-Dimethylamino-1,2-Propanediol (of formula (3))

1,2-Isopropylidene-sn-glycerol 3-tosylate (10 g) was placed in a Parrpressure reactor and the entire apparatus was cooled to 0° C.Dimethylamine (approx. 10 ml) was condensed into the reactor and thevessel was sealed. The mixture was heated under pressure for 2 days. Thereaction vessel was cooled to 0° C. and opened. The excess dimethylaminewas allowed to evaporate and the residue was dissolved in methanol (100ml) containing concentrated hydrochloric acid of pH 3, and the mixturewas heated at reflux for 2 hours. After removal of the solvent in vacuo,the residue was partitioned between concentrated NaOH (5 ml, 10M) andtetrahydrofuran (100 ml). The tetrahydrofuran layer was evaporated toafford the title compound as a pale yellow oil.

[α]_(D) ²⁵=−26.8° (1% CH₃CO₂H/H₂O); ′H NMR (90 MHz, CDCl₃ δ 4.0-3.3 (m,3H), 2.9 (OH), 2.8-2.0 (m, 8H).

Preparation 2N-(2,3-Di-(9-(Z)-octadecenyloxy))prop-1-yl-N,N,-dimethylamine (offormula (4))

In accordance with Reaction Scheme I, a mixture of3-(dimethylamino)-1,2-propanediol (1.19 g, 10 mmol), potassiumtert-butoxide (3.36 g, 30 mmol) and oleyl toluenesulfonate (12.7 g, 30mmol) in xylenes (50 ml) was stirred at room temperature under housevacuum (approx. 30 torr) for 0.5 hour. The mixture was heated to 50° C.and stirred for an additional 0.25 hour. The vacuum was removed and thereaction vessel was filled with nitrogen gas to room pressure (approx. 1atm.). The temperature was increased until the reaction boiled(approximately 140° C.) and the mixture was stirred at reflux for 3hours. The mixture was diluted with hexane (100 ml) and extracted withwater (2×50 ml). The organic layer was concentrated, applied to a columnof silica gel (150 g) packed in hexanes in ether (1:2), then eluted withthe same solvent mixture to give the title compound (4.5 g) as an oil.

Proceeding in a similar manner, but substituting for3(dimethylamino)-1,2-propanediol the appropriate precursor, thefollowing compounds were made:

(±) N-methyl-N-(2,3-di-hexadecyloxy)-prop-1-yl-pyrrolidine;

(±) N-methyl-N-(2,3-di-hexadecyloxy)-prop-1-yl-piperidine;

(±) N-methyl-N-(2,3-di-hexadecyloxy)-prop-1-yl-morpholine; and

(S)-N-(2,3-Di-(9-(Z)-octadecenyloxy))prop-1-yl-N,N,-dimethylamine.

Proceeding in a similar manner, but substituting for thep-toluenesulfonate of oleyl alcohol the appropriate alkylating agent,the following compound was made:

(±)N-(2,3-dihexadecyloxy)-prop-1-yl-N,N-dimethylamine.

Proceeding in a similar manner, but substituting for thep-toluenesulfonate of oleyl alcohol the appropriate precursor, thefollowing compound is made:

N-(2,3-di-(dec-2-ynyloxy))-prop-1-yl-N,N-dimethylamine.

Preparation 3 1,2:3,4:5,6-Triisopropylidine-D-Mannitol (of formula (6))

Perchloric acid (3.5 ml, 70%) was added to a mixture of D-mannitol (100g) and 2,2-dimethoxypropane (700 ml) in acetone (100 ml). After stirringthis mixture at room temperature for 18 hours, sodium bicarbonate (5 g)was added to the solution. This mixture was stirred at room temperaturefor 1 hour and then filtered. The filtrate was concentrated to ½ of theoriginal volume and diluted with water (500 ml) to give the titlecompound.

Preparation 4 D-Mannitol-3,4-Acetonide (of formula (7))

1,2:3,4:5,6-Triisopropylidine-D-Mannitol (90 g) was dissolved in 70%acetic acid (250 ml) and heated at 45° C. for 1.5 hours. The mixture wasconcentrated in vacuo to an oil. This oil was resuspended in toluene(150 ml) and again concentrated in vacuo. The resulting oil wasdissolved in ethylacetate (400 ml) and cooled to −5° C. The titlecompound crystallized from this mixture.

Preparation 5 1,2,5,6-Tetraoleyl-D-mannitol-3,4-acetonide (of formula(8))

D-Mannitol-3,4-acetonide (5.0 g, 22.52 mmol) was dissolved indimethylformamide (200 ml, distilled from calcium hydride under reducedpressure). To this solution was added sodium hydride (1.08 grams, 22.52mmol, 50% oil dispersion) and the mixture was heated to 50° C. andstirred for 1 hour (mechanical stirrer required) To the resultingmixture was added the toluenesulfonate of oleyl alcohol (9.5 grams,22.52 mmol). The temperature was increased to 90° C. and stirring wascontinued for 1 hour.

The sequence of addition of sodium hydride (same amount) and stirring 1hour, then addition of oleyl tosylate (same amount) and stirring 1 hour,all at a constant 90° C., was repeated 4 more times (total of 5 times).The reaction mixture was allowed to cool to room temperature than pouredslowly into a saturated solution of sodium chloride (500 ml). Theresulting mixture was extracted with hexanes (3×250 ml), dried(potassium carbonate) and concentrated. The crude product waschromatographed over silica gal (1000 grams) eluting with a gradient offrom 0 to 5% diethyl ether in hexanes to give 13.93 grams of the titlecompound as a viscous oil.

Proceeding in a similar manner, but substituting for thetoluenesulfonate of oleyl alcohol the appropriate precursor, thefollowing compounds were made:

1,2,5,6-tetradecyl-D-mannitol-3,4-acetonide;

1,2,5,6-tetrahexadecyl-D-mannitol-3,4-acetonide;

1,2,5,6-tetradocosyl-D-mannitol-3,4-acetonide;

1,2,5,6-tetra-(4-Z-decenyl)-D-mannitol-3,4-acetonide.

Preparation 6 1,2,5,6-Tetraoleyl-D-mannitol (of formula (9))

To a solution of 1,2,5,6-tetraoleyl-D-mannitol-3,4-acetonide (24.0grams, 19.62 mmol) in tetrahydrofuran (100 ml) was addedH₂O:trifluoroacetic acid (1:9, 100 ml). This solution was stirred for 1hour at 50° C., then concentrated to an oil by rotary evaporation.Toluene (200 ml) was added and evaporated to azeotropically remove theresidual acid. The crude material was dissolved in diethyl ether (100ml) and a saturated solution of ammonium hydroxide in water (10 ml) wasadded. This mixture was stirred for 2 hours and then the ether phase waswashed two times with water, dried (magnesium sulfate) and concentrated.The crude product was suitable for further reaction; a small portion waspurified by column chromatography over silica gel (10% ethylacetate/hexanes) to give an analytical sample of the desired diol as aviscous oil.

Proceeding in a similar manner, but substituting for the1,2,5,6-tetraoleyl-D-mannitol-3,4-acetonide of formula (8) theappropriate precursor, the following compounds were made:

1,2,5,6-tetradecyl-D-mannitol;

1,2,5,6-tetrahexadecyl-D-mannitol;

1,2,5,6-tetradocosyl-D-mannitol;

1,2,5,6-tetra-(4-Z-decenyl)-D-mannitol; and

1,6-didecyl-2,5-dihexadecyl-D-mannitol.

Preparation 7(S)-N-(2,3-Di-(9-(Z)-octadecenyloxy))prop-1-yl-N,N,-dimethylamine (offormula (11))

The crude diol 1,2,5,6-tetraoleyl-D-mannitol described in the previousexample, was dissolved in chloroform (500 ml) and lead tetraacetate(11.8 g, 26.0 mmol) was added. This mixture was stirred for 2 hours andthen ethylene glycol (5 ml) was added followed quickly by water (100ml). The water phase was drawn off and the organic phase was washed oncewith saturated sodium chloride solution, dried (magnesium sulfate), andconcentrated to an oil to give the crude aldehyde of formula (10) whichwas used immediately in the next step.

To a solution of dimethylamine hydrochloride (35.5 g, 435 mmol) inmethanol (150 ml) was added anhydrous sodium acetate (24 g, 282 mmol).The mixture was stirred for 1 hour and then the resulting sodiumchloride was filtered off and the clear methanol solution added to thecrude aldehyde. Tetrahydrofuran (150 ml) was added followed by 3angström molecular sieves (about 20 g). Sodium cyanoborohydride (1.5 g,23.9 mmol) was added and the mixture was stirred at 50° C. for threedays. The crude reaction mixture was filtered through Celite (washingwith 1:1 tetrahydrofuran) and the solution was strongly acidified with1N HCl and stirred for ½ hour. The solution was then made strongly basicwith 10 NaOH and extracted with diethyl ether (3×200 ml). The crudeproduct was purified by column chromatography over silica gel using agradient of 0 to 10% methanol in chloroform to give the captioneddimethylamino product as a viscous oil.

The hydrochloride salt of the title compound was prepared by dissolvingthe foregoing product (100 mg) in ether (10 ml) and adding three dropsof ethyl acetate saturated with HCl gas. The resulting solution wasconcentrated and placed under high vacuum for 24 hours. The resultingproduct was a gummy solid.

Proceeding in a similar manner, but substituting for the1,2,5,6-tetraoleyl-D-mannitol of formula (9) the appropriate precursor,the following compounds are made:

(S) N-(2,3-di-decyloxy)-prop-1-yl-N,N-dimethylamine;

(S) N-(2,3-di-hexadecyloxy)-prop-1-yl-N,N-dimethylamine;

(S) N-(2,3-di-(4-(Z)-decenyloxy))-prop-1-yl-N,N-dimethylamine; and

(S) N-(2,3-di-docosyloxy)-prop-1-yl-N,N-dimethylamine.

Preparation 8 2,5-Di-O-benzyl-1,3:4,6-di-O-benzylidene-D-mannitol (offormula (13))

Powdered potassium hydroxide (37 g) was added to1,3:4,6-di-O-benzylidene-D-mannitol (10 g) dissolved in benzyl chloride(64 ml). The mixture was heated at 140° C. for 3 hours, then cooled anddiluted with water (200 ml). Extraction with chloroform, followed bywashing with water and evaporation gave a solid, which was crystallizedfrom petroleum ether to give the captioned compound, m.p. 102-3° C.

Preparation 9 2,5-Di-O-benzyl-D-mannitol (of formula (14))

2,5-Di-O-benzyl-1,3:4,6-di-O-benzylidene-D-mannitol (10.9 g) dissolvedin ethanol (150 ml) and water (22 ml) was treated with 1M HCl (7 ml).After refluxing this mixture for 4.5 h, the reaction was cooled andquenched with barium carbonate, then evaporated to dryness. The solidresidue was triturated with hot ethyl acetate, which was then evaporatedto give the captioned compound, m.p. 116-117° C.

Preparation 10 2,5-Dibenzyl-D-mannitol-3,4-acetonide (of formula (15))

2,5-Dibenzyl-1-mannitol (48 g, 133 mmol) dissolved in dry acetone (100ml) was treated with copper(II)sulfate (10 g, 62.6 mmol) andconcentrated sulfuric acid (2 ml). After stirring at room temperaturefor 48 hours, the mixture was quenched by the addition of solid sodiumcarbonate, followed by stirring for 3 hours. The reaction mixture wasfiltered and concentrated and the residue was crystallized from hexaneethyl acetate to give 38.0 g of the title compound, m.p. 73-74° C.

Preparation 11 2,5-Dibenzyl-1,6-didecyl-D-mannitol-3,4-acetonide (offormula (16))

A mixture of 2,5-dibenzyl-D-mannitol-3,4-acetonide (100 g, 25 mmol),powdered potassium hydroxide (23 g) and decyl bromide (40 ml) in xylene(300 ml) was heated at reflux for 4 hours. The mixture was cooled,diluted with hexanes (300 ml), decanted from excess salts and applied toa column of dry silica gel (1 Kg). Elution with hexanes followed by agradient of 0 to 50% ether in hexanes gave the title compound as an oil.

Preparation 12 1,6-Didecyl-D-mannitol-3,4-acetonide (of formula (17))

Dibenzyl compound of Example 6 (6.0 g, 8.8 mmol) was dissolved intetrahydrofuran/methanol (1:1, 100 ml). After bubbling nitrogen throughfor several minutes, 10% palladium on carbon (1 gram) was added and themixture was shaken at 70° C. under 60 psi hydrogen for 48 hours. Themixture was filtered and concentrated to give the title compound (4.3 g)as a white solid; m.p. 36-39° C.

Preparation 13 1,6-Didecyl-2,5-dihexadecyl-D-mannitol-3,4-acetonide (offormula (18))

1,6-Didecyl-D-mannitol-3,4-acetonide (4.3 g, 8.57 mmol) andbromohexadecane (7.84 g, 25.7 mmol) were dissolved in xylene (40 ml) andKOH (5.0 grams) was added. This mixture was stirred at reflux for 1.5hours. After cooling the mixture was decanted onto a column of silicagel (dry, 200 g), then eluted with hexanes followed by 3% ether inhexanes to give the title compound (7.3 g) as an oil.

Preparation 14 1,2,10-Decanetriol (of formula (20))

9-Decen-1-ol (25.0 g, 160 mmol) was dissolved in a solution made up oft-butanol (100 ml), acetone (90 ml) and water (10 ml. To this solutionwas added trimethylamine-N-oxide (26.6 g, 240 mmol) and 2 ml of asolution of osmium tetroxide (500 mg) in t-butanol (25 ml). Theresulting solution was stirred 20 hours under nitrogen then 10% sodiumbisulfite was added (50 ml). The mixture was concentrated, then taken upin trichloromethane and washed 2 times with water, dried with Na₂SO₄ andconcentrated to give 1,2,10-decanetriol as an oil. This material wasused without further purification in preparation of the correspondingacetonide (of formula 21).

Preparation 15 1,2,6-Hexanetriol-1,2-acetonide (of formula (21))

1,2,6-Hexanetriol (31 g, 0.23 mmol) was stirred with acetone (150 ml).To this mixture was added concentrated sulfuric acid (5 drops). Theresulting solution was stirred for 2 hours at room temperature. Thereaction solution was diluted with diethyl ether, washed with saturatedsodium bicarbonate solution, dried (magnesium sulfate) and concentratedto give the title compound (31 g) as a clear oil.

Proceeding in a similar manner, but substituting for the1,2,6-hexanetriol the appropriate precursor, the following compoundswere made:

1,2,4-butanetriol-1,2-acetonide;

1,2,10-decanetriol-1,2-acetonide.

Preparation 16 1,2-Hexanediol-1,2-acetonide-6-allyl ether (of formula(22))

The acetonide of Preparation 15 (30 g, 172 mmol) was dissolved in drydimethylformamide (500 ml). To this solution was added sodium hydride(8.28 g, 172 mmol, 50 oil dispersion) and the mixture was stirred for ½hour at room temperature then warmed to 90° C. over ½ hour. To thismixture was added allyl chloride (21 ml, 258 mmol) and the stirring wascontinued for 1 hour. After cooling, the mixture was poured into waterand extracted with ether (2×100 ml). The combined ether extracts werewashed with brine, dried (magnesium sulfate) and concentrated.Chromatography over silica gel (10% ether in hexanes) gave the titleproduct as a clear oil; bp=70° C. at 0.001 mmHg.

Proceeding in a similar manner, but substituting for1,2,6-hexanetriol-1,2-acetonide the appropriate precursor, the followingcompounds were made:

1,2-butanediol-1,2-acetonide-4-allyl ether;

1,2-decanediol-1,2-acetonide-10-allyl ether.

Preparation 17 1,2-di-hydroxy-hexan-6-allyl ether (of formula (23))

In ethanol (100 ml) was dissolved 1,2-hexanediol-1,2-acetonide-6-allylether (20 g, 93.9 mmol) to which was added 20 ml of 1N HCl. The solutionwas then heated to 50° C. for 2 hours. The resulting solution wasconcentrated, then taken up in chloroform (100 ml) and washed with brine(2×10 ml, dried (sodium sulfate) and concentrated to give the titlecompound as a clear oil.

Proceeding in a similar manner, but substituting for1,2-hexanediol-1,2-acetonide-6-allyl ether the appropriate precursor,the following compounds were made:

1,2-di-hydroxy-butan-4-allyl ether

1,2-di-hydroxy-decane-10-allyl ether.

Preparation 18 1,2-Di(9-(Z)-octadecenyloxy)-hexan-6-allyl ether (offormula (24))

The diol of Preparation 17 (3.45 g, 19.83 mmol) was dissolved in drydimethylformamide (60 ml). To this solution was added sodium hydride(951 mg, 19.5 mmol). The mixture was heated to 90° C. and oleyl tosylate(8.37 g, 19.8 mmol) was added. Stirring was continued for 1 hour atwhich time a second equivalent of sodium hydride (951 mg, 19.8 mmol) wasadded. After 15 minutes a second equivalent of oleyl tosylate (8.37 g,198. mmol) was added and stirring was continued for I hour. The reactionmixture was poured into water and extracted with ether (2×100 ml).Column chromatography over silica gel (0 to 5% ether/hexanes) gave 3.5 gof the title compound as a clear oil.

Proceeding in a similar manner, but substituting for5,6-di-hydroxy-hexan-1-allyl ether the appropriate precursor, thefollowing compounds were made:

1,2-di(9-Z-octadecenyloxy)-butan-4-allyl ether;

1,2-di(9-Z-octadecenyloxy)-decan-10-allyl ether.

Preparation 19 1,2-Di(9-(Z)-octadecenyloxy)-hexan-6-ol (of formula (25))

The triether of Preparation 18 (3.20 g, 4.74 mmol) was dissolved inethanol/tetrahydrofuran (1:1, 30 ml) and Wilkinsons catalyst(tris(triphenylphosphine)rhodium chloride, 200 mg) was added followed by0.1N HCl (1 ml). This mixture was refluxed for 3 hours then 1 N HCl (5ml) was added and refluxed 4 hours. The solution was cooled andconcentrated. Diethyl ether was added and washed with brine, dried(magnesium sulfate), concentrated and chromatographed over silica gel (5to 50% ether in hexanes) to give 2.56 g of the title alcohol as an oil.

Proceeding in a similar manner, but substituting for1,2-di(9-Z-octadecenyloxy)-6-allyloxyhexane the appropriate precursor,the following compounds were made:

1,2-di(9-Z-octadecenyloxy)butan-4-ol;

1,2-di(9-Z-octadecenyloxy)decan-10-ol.

Preparation 20 (%)N-(5,6-di-(9-(S)-octadecenyloxy))-hex-1-yl-N,N-dimethyl amine (offormula (27))

The substituted hexan-6-ol from Preparation 19 (2.50 g, 3.94 mmol) wasdissolved in pyridine (20 ml) and p-toluenesulfonyl chloride (0.90 g,4.73 mmol) was added. This mixture was stirred overnight at roomtemperature then poured into ice water and stirred ½ hour. The resultingmixture was extracted with ether and the ether phase was washed with0.1N HCl, dried (magnesium sulfate) and concentrated. This crudeintermediate was immediately dissolved in dimethylamine and placed in asealed tube at room temperature for 20 hours. The tube was cooled to 0°C. and opened. The dimethylamine was allowed to evaporate under a streamof nitrogen. Column chromatography of the crude product over silica gel(0 to 5% methanol in chloroform) gave the title product as a very thickoil. The hydrochloride was prepared as described in Preparation 7. Thiswas also an oil, NMR (300 MHz, CDCl₃) 5.40-5.30 (m, 4H), 3.65-3.50 (m,1H), 3.50-3.30 (m, 6H), 3.05-2.90 (m, 2H), 2.79 (s, 6H), 2.10-1.65 (m,11H), 1.65-1.45 (m, 8H), 1.45-1.15 (m,44H), 0.95-0.80 (m, 6H).

In a similar manner, but substituting the appropriate starting material,the following compounds were prepared:

(±) N-(3,4-di-(9-(Z)-octadecenyloxy))-but-1-yl-N,N-dimethylaminehydrochloride, oil, NMR (90 MHz, CDCl₃) 5.33 (t, J=5 Hz, 4H), 3.85-3.15(m, 18H), 2.20-1.80 (m, 8H), 1.70-1.00 (m, 50H), 0.88 (t, J=7 Hz, 6H);

(±) N-(9,10-di-(9-(Z)-octadecenyloxy))-dec-1-yl-N,N-dimethylaminehydrochloride, wax, NMR (90 MHz, CDCl₃) 5.34 (t, J=5 Hz, 4H), 4.65-4.25(m, 9H), 2.81 (s, 3H), 2.75 (s, 3H), 2.20-1.75 (m, 8H), 1.75-1.00 (m,62H), 0.88 (t, J=7 Hz, 6H).

Preparation 21 2,3-Di(9-(Z)-octadecenyl)propan-1-ol (of formula (25))

Crude 2,3-di(9-(Z)-octadecenyl)propanal of formula (10) from Preparation7 (10.0 g, 16.9 mmol) was dissolved in tetrahydrofuran/methanol (1:1,200 ml) and cooled to 0° C. Sodium borohydride (3.13 g, 85.0 mmol) wasadded and the mixture was stirred overnight. The solution was acidifiedwith 1N HCl to pH<2, diluted with ether, washed with water, concentratedand column chromatographed (chloroform) to give the title compound as anoil.

Preparation 22 1,2-Di(9-(Z)-octadecenyloxy)-3-iodopropane (of formula(26))

The alcohol 2,3-di(9-(Z)-octadecenyl)propan-1-ol (5.0 g, 8.36 mmol) wasdissolved in pyridine (50 ml) and p-toluenesulfonxyl chloride (1.91 g,10.0 mmol) was added. The solution was stirred for 24 hours then pouredinto ice water, extracted with ether and washed with 1N HCl until theaqueous layer remained acidic. The organic phase was dried (magnesiumsulfate) and concentrated to give crude tosylate. The material wasdissolved in methyl ethyl ketone (50 ml), sodium iodide (1.5 g, 10.0mmol) was added and the solution was refluxed for 5 hours. The solventwas removed and the residue was taken up in ether and washed with water.The organic layer was concentrated and chromatographed to give the titlecompound as an oil.

EXAMPLE 1 (S)N-(2,3-Di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride

The dimethylamino productN-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N-dimethylamine (10 grams)was placed in a Parr pressure reactor and cooled to −78° C. Methylchloride (about 50 ml) was condensed into the reaction vessel, which wasthen sealed and heated to 70° C. for 48 hours. The reaction vessel wascooled and opened and the methyl chloride allowed to evaporate under astream of nitrogen. The crude product was crystallized from acetonitrileto give the title compound as an off-white solid, (S)N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride, [α]_(D) ²⁵−20.0° (CHCl₃);

Proceeding in a similar manner, but substituting forN-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N-dimethylamine theappropriate precursor, the following compounds are prepared:

(S) N-(2,3-di-decyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride, m.p.87-88° C., [α]_(D) ²⁵−26.5° (CHCl₃);

(S) N-(2,3-di-hexadecyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride,[α]_(D) ²⁵−23.4° (CH₃OH);

(S) N-(2,3-di-(4-(Z)-decenyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride, wax, [α]_(D) ²⁵ 0° (CHCl₃);

(S) N-(2,3-di-docosyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride,m.p. 161-163° C., [α]_(D) ²⁵−15.7° (CHCl₃);

(±) N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride, m.p. 35-38° C., NMR (300 MHz, CDCl₃) 5.35 (t, J=5 Hz, 4H),4.15-3.90 (m, 2H), 3.80-3.40 (m, 3H), 3.49 (s, 9H), 3.43 (t, J=7 Hz,4H), 2.01 (m, 8H), 1.56 (m, 4H), 1.27 (m, 40H), 0.88 (t, J=7 Hz, 6HO);

(±) N-(2,3-dihexadecyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride,m.p. 76-78° C.;

(±) N-methyl-N-(2,3-di-hexadecyloxy)-prop-1-yl-pyrrolidinium chloride,m.p. 71-73° C.;

(±) N-methyl-N-(2,3-di-hexadecyloxy)-prop-1-yl-piperidinium chloride,m.p. 111-116° C.;

(±) N-methyl-N-(2,3-di-hexadecyloxy)-prop-1-yl-morpholinium chloride,m.p. 118-121° C.;

(±) N-(5,6-di-(9-(Z)-octadecenyloxy))-hex-1-yl-N,N,N-trimethylamnoniumchloride, oil;

(±) N-(9,10-di-(9-(Z)-octadecenyloxy))dec-1-yl-N,N,N-trimethylammoniumchloride, oil, NMR (300 MHz, CDCl₃) 5.40-5.30 (t, J=t Hz, 4H), 3.70-3.30(m, 9H), 3.46 (s, 9H), 2.10-1.90 (m, 8H), 1.85-1.65 (m, 2H), 1.60-1.20(m, 50H), 0.88 (t, J=7 Hz, 6H);

(±) N-(3,4-di-hexadecyloxy)but-1-yl-N,N,N-trimethylammonium chloride,m.p. 177-179° C.; and

(S) N-(3-decyloxy-2-hexadecyloxy)prop-1-yl-N,N,N-trimethylammoniumchloride, m.p. 88-90° C., [α]_(D) ²⁵−24.7° (CHCl₃).

Proceeding in a similar manner, but substituting forN-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N-dimethylamine theappropriate precursor, the following compound is made:

N-(2,3-di-(dec-2-ynyloxy))-prop-1-yl-N,N,N-trimethyl-ammonium chloride.

EXAMPLE 2 (±) N-(2,3-Dihexadecyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride

Dimethyl sulfate (187 g, 1.5 moles) was added dropwise to a solution of(±) N-(2,3-dihexadecyloxy)-prop-1-yl-N,N-dimethylamine (740 g, 1.3moles) in toluene (2 L) at room temperature. After completion of theaddition, the mixture was stirred for one additional hour at roomtemperature. This solution was extracted with saturated sodium chloride(2×500 mL) and the toluene layer was diluted with acetone (2L) andcooled to 5° to give the title compound as a colorless solid, (±)N-(2,3-dihexadecyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride, m.p.76-78° C.

EXAMPLE 3 N-(2,3-Di-(9-(Z)-octadecenyloxy))-prop-1-yl-quinuclidiniumchloride (Formula I)

The iodopropane of Example 14 (2.0 grams, 2.82 mmol) was dissolved indichloromethane (1 ml) and quinuclidine (1.57 grams, 14.1 mmol) wasadded. The solution was sealed in a pressure reactor and heated to 100°C. for 48 hours. The crude product was chromatographed over a small plugof silica gel (0 to 5% methanol in chloroform) and then ion exchangedover Dowex 2-X8 (chloride form, eluting with methanol) to give the titlecompound, (S) N-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-quinuclidiniumiodide, m.p. 81-83° C., [α]_(D) ²⁵−33.5° (CDCl₃);

In a similar manner, but substituting the appropriate starting material,the following compounds are prepared:

N-(2,3-Di-(9-(Z)-octadecenyloxy))-prop-1-yl-N-ethyl-N-ethyl-N-methyl-N-phenylammoniumiodide;

N-(2,3-Di-(9-(Z)-octadecenyloxy))-prop-1-yl-N-benzyl-N,N-dimethylammoniumiodide;

N-(2,3-Di-(9-(Z)-octadecenyloxy))-prop-1-yl-N-benzyl-N,N,N-triphenylammoniumiodide;

N-(2,3-Di-(9-(Z)-octadecenyloxy))-prop-1-yl-pyridinium iodide.

EXAMPLE 4

The following compositions illustrate the use of the instant compoundsin pharmaceutical formulations.

1) Thirty-four mg ofN-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride and 6.3 mg of9-(1,3-dihydroxy-2-propoxymethyl) guaninedipalmitate were dissolved in chloroform/methanol (2:1:2 ml). Solventwas removed under a stream of nitrogen and placed in vacuo overnight.The dried film was suspended in 1 ml of 50 mM phosphate buffered saline,pH 7.4 and sonicated to clarity.

2) A topical formulation was prepared by dissolving 0.025 mg offluocinolone acetonide [6α, 9α-difluoro-11β, 16α, 17α,21-tetrahydroxypregna-1,4-diene-3,20-dione 16,17-acetonide] 0.25 gramsof N-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethyl ammoniumchloride in 20 ml of dichloromethane. The solvent was evaporated under astream of nitrogen gas until a dry film was obtained. The film mixturewas placed under vacuum overnight to evaporate off the dichloromethanecompletely. The dry film was then suspended in 25 ml of 1% sodiumchloride solution. The suspension was sonicated until visually clear.

3) Fluocinonide [6α, 9α-difluoro-11β, 16α, 17α,21-tetrahydroxypregna-1,4-diene-3,20-dione, 16,17-acetonide-21-acetate]0.025 grams and 1.0 grams ofN-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride were dissolved in 20 ml of dichlormethane which was thenevaporated under a stream of nitrogen gas until a dry film was obtained.This film mixture was placed under vacuum overnight to evaporate offresidual dichloromethane. The resulting film was suspended in 25 ml of1% sodium chloride solution and sonicated until visually clear.

4) There was dissolved 160 mgN-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride and 20 mg butoconazole nitrate[1-[4-(4-chlorophenyl)-2-(2,6-dichlorophenylthio)-n-butyl] imidazolenitrate] in 2 mls of chloroform. The chloroform was removed under astream of nitrogen and the residue was placed under vacuum overnight toremove residual chloroform. The resulting film was suspended in 2 mls ofpurified water by hand shaking and vortexing.

5) Diarachidoylphosphatidyl choline, 60 mg, and 5.4 mgN-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride was dissolved in 2 ml of chloroform which was removed under astream of nitrogen and placed under vacuum overnight to remove residualsolvent. The resulting film was suspended in 2 mls of purified watercontaining 20 million units of beta-interferon by gentle trituration toavoid excessive foaming.

6) Thirty mg ofN-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride and 3 mg of6-O-stearoyl-N-acetyl-muramyl-L-α-aminobutyryl-D-isoglutamine weredissolved in chloroform. A nitrogen stream was used to remove themajority of the solvent, the residual solvent being removed undervacuum. The resulting film was suspended in 1 ml of purified water andtreated with ultrasound until clear.

7) Distearoylphosphatidyl choline, 2.22 mg, 1 mgN-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride and 0.23 mg of cholesterol were dissolved in 1 ml chloroform.Solvent was removed under a stream of nitrogen and the residue placedunder vacuum overnight. The dried film was suspended in 6 mM phosphatebuffered saline containing 8% Triton X-100 (0.5 ml). To this was added50 μg of lectin affinity column purified bovine herpes antigen. Then 1ml of packed BioBeads was added (to remove Triton X-100) and shakengently for 2 hours at 55° C. after which the BioBeads were decanted.

8) 14 Micromoles of dioleoylphosphatidyl choline and 6 micromoles ofN-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride were dissolved in 2 ml of chloroform and then dried down undera stream of nitrogen. The resulting dried film was placed under vacuumfor ½ hour, after which the film was then dissolved into 1 ml ofcyclohexane, transferred to a 100 ml round-bottomed flask and frozen ondry ice. The flasks were then attached to a lyophilization apparatus andthe cyclohexane removed. Murine gamma interferon solution [0.2 ml(500,000 units/ml) was suspended in a buffer containing 10 mMmonopotassium phosphate, 2 mM sodium chloride and 3 mM potassiumchloride (adjusted to pH 8.0 with potassium hydroxide)] was then addedto the lyophilized lipids which caused formation of liposomes. Theliposomes were then diluted to a convenient concentration with morephosphate buffer as needed.

In a similar manner other concentrations of drug in liposomes can beprepared. By varying the amount of aqueous solution added to the film,the concentration of drug in the final liposome formulation can bevaried between 0.05 and 10% by weight.

9) Two types of lipid were used and compared in the presence of threedifferent buffers.

Lipids:

1) DOTMA; 13 mg

2) DOPG/DOPC, 7.3 (w/w); 13 mg

Buffers:

1) 0.02 M phosphate; 0.9% NaCl

2) 0.02 M isotonic glucose

3) deionized water

Drug: 0.9 mg ketorolac free acid

Lipid stock solutions were prepared in chloroform at 20 mg/ml. A 0.01 Mstock solution of ketorolac was prepared in methanol. The lipid and drugwere mixed into a glass vial and the solvent was removed under a streamof nitrogen gas. The dried films were evacuated overnight on a vacuumpump to remove traces of solvent. Each of the drug/lipid films wasreconstituted by vortexing with one of the buffers. 0.15 μl of theliposome suspension was removed and centrifuged at 100,000×g in aBeckman Airfuge. Only the liposome formulations containing NaCl(buffer 1) gave pellets after centifugation. The supernatants werewithdrawn from these preparations and assayed in a DV spectrophotometer(peak absorbance 319 nm). The results indicated that the DOTMA liposomesentrapped more than 80% of the drug, whereas, the conventional DOPC/DOPGliposome entrapped less than 5% of the drug.

Another DOTMA/ketorolac formulation was prepared identical to the above,except that 0.1 mg of the rhodamine phosphatidyl ethanolamine (inchloroform) was added to the mixture before solvent removal undernitrogen. The resulting fluorescent liposomes were applied to dissectedrabbit cornea for 2 hours, washed with isotonic saline, and prepared forcryostat sectioning. Thin sections were cut and observed by fluorescentmicroscopy. Fluorescence was observed on the surface of the corneaindicated the liposomes adhered to rabbit cornea.

EXAMPLE 5 Intracellular Introduction of Oligoribonucleotides

Oligoadenylates of the general structure ppp(A2′p)_(n)A, n≧2, arereferred to as “2-5A”. 2-5A molecules are well known, to those skilledin the art, to bind to an intracellular endonuclease resulting inactivation of the endonuclease with subsequent cleavage of ribosomal andmessenger RNA, resulting in inhibition of protein synthesis. However dueto the highly negatively-charged nature of the 2-5A oligonucleotides,they do not cross the intact cell membrane and thus are inactive whenadded to intact, nonpermeabilized cells. A calcium phosphatecoprecipitation technique can be employed to artificially introduce 2-5Ainto cells, which can be monitored by inhibition of protein synthesis.Thus 10⁻⁵ to 10^(−9 M) 2-5A was added to a solution of 120 mM CaCl₂,which was then added dropwise into bubbled Hepes buffered saline, pH7.05 (8.0 g NaCl, 0.37 g KCl, 0.125 g Na₂HPO₄″2H₂O, 1.0 g glucose, and5.0 g Hepes per liter). A final sample volume of 0.5 ml was added toquadruplicate wells of Costar 24 well plates containing confluent Veroor L₉₂₉ cells. After 45 minutes at room temperature, an additional 0.5ml of media was added to each well and incubated an additional 90minutes at 37° C. The solutions were then removed by aspiration, thecells were labelled for 90 minutes with [³H] leucine (1 μCi/0.5ml/well), and the trichloroacetic acid (TCA)-precipitable radioactivitywas determined after incubating 30 minutes with cold 10% TCA, washing,harvesting the cells with 0.1 M NaOH, neutralizing with HCl, andquantitating the radioactivity by liquid scintillation counting. Theresults show that ppp(A2′p)₂A, after the calcium-phosphate precipitationtechnique, resulted in a dose-dependent inhibition of protein synthesis.50%-maximal inhibition occurred at approximately 10⁻⁹ M 2-5A. However,the calcium phosphate treatment was very toxic to the cells, asdetermined from their morphology as well as approximately up to 40%reduction in protein synthesis of the cells receiving only the calciumphosphate treatment but no 2-5A. Cells that received all concentrationsof 2-5A without addition of calcium phosphate coprecipitation showed noinhibition of protein synthesis.

DOTMA liposomes were prepared by drying DOTMA solutions in CHCl₃ to afilm under a stream of nitrogen, removing residual solvent by placingunder vacuum overnight, and then suspending in water with vigorousshaking and then sonicated in a bath type sonicator for 30 minutes.DOTMA liposomes were added to Vero cells in 24 well plates to give afinal concentration of 0.015, 0.03, 0.06, or 0.12 mM DOTMA. Cells wereincubated and protein synthesis was determined as described above exceptthat the 60 mM CaCl₂ was not included. No significant inhibition ofprotein synthesis was obtained after 2 hours incubation. In anothertest, cells were incubated with samples for 2, 6, or 18 hours andprotein synthesis was determined as described above. Samples were alsoincluded that contained a lipophilic derivative of 2-5A[5′-triphosphophoryladenylyl-(2′→5′)adenylyl-(2′→5′)adenylyl-(2′→6′)(3′-aza-N-hexyl-1′,2′,3′,4′-tetradeoxyhexopyranos-1′-yl)adenine] prepared in the DOTMA liposomes in thefollowing ratios:

0.03 mM DOTMA: 10⁻⁶ M 2-5A analog;

0.06 mM DOTMA: 10⁻⁶ M 2-5A analog;

0.12 mM DOTMA: 10⁻⁶ N 2-5A analog.

When these preparations containing the 2-5A analog formulated in DOTMAliposomes were added to the cells, a significant inhibition of proteinsynthesis was obtained. After 6 hours incubation, the 2-5A analog aloneat 10⁻⁶ M gave 82±14% of protein synthesis of untreated cells, Danaalone at 0.015, 0.03, and 0.06 mM gave 86±8, 80±6, and 77±4% of proteinsynthesis of untreated cells, whereas calcium phosphate alone gave 64±3%of protein synthesis of untreated cells. 10⁻⁶ M 2-5A analog formulatedin Dana at 0.015, 0.03, or 0.06mM gave 23±7, 41±10, and 17±2% of proteinsynthesis of untreated cells. Thus the DOTMA formulation of the 2-5Aanalog served to introduce the 2-5A analog intracellularly, resulting ininhibition of cellular protein synthesis.

EXAMPLE 6

The ability to solubilize a drug is increased by use of the compoundsand liposomes of this invention. In this way a greater concentration ofa normally insoluble or sparingly soluble drug can be presented to thebody.

For example, 1-[4-(4-chlorophenyl)-2-(2,6-dichlorophenylthio)-n-butyl]imidazole nitrate without the presence of any of the compounds of thisinvention is insoluble in aqueous buffer (phosphate buffered saline, pH7.4).

A 0.3% soluble preparation of the above drug was prepared using thefollowing method:

18 mg of 1-[4-(4-chlorophenyl)-2-(2,6-dichlorophenylthio)-n-butyl]imidazole nitrate and 482 mg ofN-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride were dissolved in methylene dichloride. The methylenedichloride was evaporated under a stream of nitrogen and the dried filmplaced under vacuum overnight. The dried film was suspended in 6 mlphosphate buffered saline, pH 7.4, and sonicated to clarity.

EXAMPLE 7 DOTMA/DNA complexes

This Example illustrates the formation of complexes between theliposomes made from compounds of Formula I and DNA.

DOTMA/DOPC vesicles were prepared by dissolving 10 mg DOTMA and 10 mgDOPC in chloroform and removing the solvent by drying under a stream ofnitrogen gas. This dried film was placed under vacuum overnight toremove traces of solvent. The film was then suspended in 1 ml water withvigorous shaking and sonicated in a bath type sonicator for 30 minutesuntil clear producing DOTMA/DOPC SUV's at a concentration of 20 mg/mltotal lipid.

Calf thymus DNA (obtained from Sigma Chemical Co.) was suspended in 5 mlwater at a concentration of 1 mg/ml and sonicated in a bath sonicatorfor 30 min in order to reduce the size of the DNA strands.

The DNA was diluted with water to a concentration of 0.2 mg/ml. Liposomedilutions were made to give concentrations of 0.2, 0.4, 0.8, 1.2, 1.6,and 2.0 mg/ml lipid. Equal volumes of the DNA stock solution and each ofthe lipid dilutions were mixed and observed for the appearance ofaggregates. There was no aggregation in the samples with the highest andlowest lipid concentration. The samples at lipid dilutions of 0.8 and1.2 mg/ml yielded large macroscopic aggregates. The 0.4 and 1.6 lipiddilutions gave turbid suspensions without microscopic aggregation orsettling. Liposomes prepared without DOTMA or other compounds of FormulaI did not form precipitates when mixed with DNA, indicating that in suchcases a liposome/DNA complex did not form.

These results indicate that liposomes composed of DOTMA form a complexwith DNA and that the precise nature of this complex is dependent on themolar ratio of DNA and DOTMA liposomes added.

EXAMPLE 8 Complex Absorption to Tissue Culture Cells

This Example demonstrates the interaction of DNA/DOTMA liposomecomplexes with tissue culture cells.

DOTMA/DOPC and DOTMA/DOPE vesicles containing rhodaminephosphatidylethanolamine were prepared by dissolving 10 mg DOTMA and 0.2mg rhodamine phosphatidylethanolamine, with either 10 mg DOPC or 10 mgDOPE in chloroform and removing the solvent by drying under a stream ofnitrogen gas. These dried films were placed under vacuum overnight toremove traces of solvent. The films were then suspended in 1 ml waterwith vigorous shaking and sonicated in a bath type sonicator for 30minutes until clear producing DOTMA/DOPC and DOTMA/DOPE vesiclescontaining rhodamine-PE at a concentration of 20 mg/ml total lipid.

Calf thymus DNA (obtained from Sigma Chemical Co.) was suspended in 5 mlof water at a concentration of 1 mg/ml and sonicated in a bath sonicatorfor 30 min in order to reduce the size of the DNA strands.

The DNA was diluted to a concentration of 0.1 mg/ml and each of theliposome preparations was diluted to a concentration of 0.6 mg/ml. 0.1ml of the diluted DNA was added to 0.1 ml aliqotes of each of thediluted fluorescent liposome preparations. The samples were mixed togive the fluorescent DNA/liposome complexes.

Microscope slides containing 22×22 mm wells were seeded with 200,000mouse fibroblast cells in each well. After 24 hrs the cells were washedwith PBS and 1 ml of PBS was added to each well. 0.2 ml of each of thefluorescent liposome/DNA complexes was added to separate individualwells, the cells were incubated for 1 and 4 hours and observed, afterwashing, by fluorescence microscopy.

Based on the observed intense cell associated fluorescence it wasapparent that the DNA/Dana liposome complexes readily and extensivelyassociated with the cells. Negatively charged vesicles prepared fromDOPC:DOPG, 7:3, or neutral vesicles, prepared from DOPC, and which donot contain DOTMA, do not readily adsorb to cells.

There was an additional difference between the cells exposed tocomplexes containing DOPC and those containing DOPE. WithDOPC-containing complexes the majority of the fluorescence was punctateand associated with the cell surface. With the DOPE containingcomplexes, the fluorescence was clearly associated with the interior ofthe cell and after 4 hours there was an obvious ring of red fluorescencearound the cell nucleus.

These results indicate that the DNA/DOTMA liposome complexes associatewith tissue culture cells and that the precise way in which thecomplexes interact is critically dependent on both the presence of acompound of Formula I, as well as the other lipid component (DOPE vs.DOPC). DOPE showed special properties enabling the fluorescentcomponents of the complex to enter the cellular cytoplasm and accumulatearound the nucleus. This property can be advantageous for DNA deliveryinto cells.

EXAMPLE 9 Plasmid Transfection with DNA/DOTMA Liposome Complexes

This Example demonstrates a method for efficiently introducing andexpressing plasmids in eukaryotic cells.

DOTMA/DOPC and DOTMA/DOPE vesicles were prepared by dissolving 10 mgDOTMA, with either 10 mg DOPC or 10 mg DOPE in chloroform and removingthe solvent by drying under a stream of nitrogen gas. These dried filmswere placed under vacuum overnight to remove traces of solvent. Thefilms were then suspended in 1 ml water with vigorous shaking andsonicated in a bath type sonicator for 30 minutes until clear, producingDOTMA/DOPC and DOTMA/DOPE SUV's at a concentration of 20 mg/ml.

The pSV2CAT plasmid was grown in E. coli (obtained from BRL, BethesdaResearch Laboratory, Gaithersburg, Md., 20877), isolated and purified byprocedures commonly known in the art and stored frozen in water at aconcentration of 0.5 mg/ml. 0.05 ml of the plasmid was diluted into0.935 ml of water and 0.015 ml of liposome stock was added to produce 1ml of the DNA/DOTMA liposome complex.

500,000 mouse L929 cells were seeded onto 60 mm plastic petri dishes intissue culture media containing 10% fetal calf serum. After 24 hours thecells were washed with PBS and 5 ml PBS was added. 1.0 ml of the freshlyprepared plasmid DNA/DOTMA liposome complex was immediately added toeach dish. The cells were incubated for 4 hours at which time 4 ml ofthe PBS was removed from each plate and replaced with media containing10% fetal calf serum. For purposes of comparison one plate of cells wastransfected by the standard calcium phosphate procedure well known tothose familiar in the art, using 125 mM CaCl₂ and 7.5 mM phosphate toform the DNA precipitate, and performed according to methods reported inthe literature (see, McKinnon and Graham in Microinjection and OrganelleTransplantation Techniques: Methods and Applications, Eds. Celis et al.,Academic Press, London, 1986). After 36 hours the cells were scraped offthe plate, lysed and assayed for chloramphenicol acyl transferase (CAT)activity (same reference) which is a measure of transfection efficiencyby the pSV2CAT plasmid.

The data show that DNA/DOTMA liposome complexes prepared with DOPC gavetransfection efficiencies comparable to the standard calcium phosphateprocedure. The efficiency of transfection in the complexes containingDOPE was substantially greater than that of the standard calciumphosphate procedure.

EXAMPLE 10 Double Coated DNA/Lipid Complexes

This Example demonstrates the technique of coating the positivelycharged DNA/liposome complexes with a liposome of negative charge, inorder to eliminate localized adsorption to cells.

DOTMA/DOPC SUV's were prepared by dissolving 40 mg DOTMA and 60 mg DOPCtogether in chloroform and removing the solvent by drying under a streamof nitrogen gas. This dried film was placed under vacuum overnight toremove traces of solvent. The film was then suspended in 2 ml water withvigorous shaking and sonicated in a bath type sonicator for 60 minutesuntil clear, producing DOTMA/DOPC vesicles at a concentration of 50mg/ml.

DOPG/DOPC SUV's were prepared by dissolving 40 mg DOPG and 60 mg DOPC inchloroform and drying off the solvent under a stream of nitrogen gas.This dried film was placed under vacuum overnight to remove traces ofsolvent. The film was then suspended in 2 ml water with vigorous shakingand sonicated in a bath type sonicator for 30 minutes until clear,producing DOPG/DOPC SUV's at a concentration of 50 mg/ml.

The puC8 plasmid (obtained from BRL, Bethesda Research Laboratory,Gaithersburg, Md., 20877) was grown in E. coli, isolated and purified byprocedures commonly known in the art and stored frozen in water at aconcentration of 1.0 mg/ml.

1 μl of pUC8 plasmid was added to 10 μl of the DOTMA/DOPC vesicles andthoroughly mixed by repeated passage of the sample through a 10 μlHamilton syringe; then a 58 μl aliquot of DOPG/DOPC vesicles was addedand the mixture was repeatedly passed through a 100 μl Hamilton syringe.When this preparation was run over a gel filtration column (SephacrylS-1000) 100% of the DNA migrated with a different Stokes radius then thefree untreated plasmid and the lipid fractions exactly overlapped withthe DNA. This material does not interact with tissue culture cells.

This example demonstrates that a second coating of negatively chargedlipid can be added to DNA/DOTMA liposome complexes to yield a productthat has different properties than the starting material. This procedureresults in essentially 100% entrapment of the DNA into the liposomecomplex.

EXAMPLE 11 Lectin Coupling to Double Coated DNA Complexes

This Example demonstrates the preparation of a DNA/liposome complex thatspecifically binds to selected cell types.

DOTMA/DOPC vesicles containing rhodamine-PE were prepared by dissolving20 mg DOTMA, 30 mg DOPC and 0.5 mg in chloroform and drying off thesolvent under a stream of nitrogen gas. This dried film was placed undervacuum overnight to remove traces of solvent. The film was thensuspended in 1 ml water with vigorous shaking and sonicated in a bathtype sonicator for 30 minutes until clear producing DOTMA/DOPC SUV's ata concentration of 50 mg/ml.

Maleimide-PE (chemical name: dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexanel-carboxylate) was prepared by mixing17 mg succinimidyl 4-(N-maleimidomethyl) clyclohexane-1-carboxylate(SMCC), with 7 μl triethylamine and 25 mgdioleoylphosphatidylethanolamine (DOPE) in 2.5 ml of chloroform. Themixture was sealed tightly to prevent evaporation and stirred at roomtemperature overnight. The reaction mixture was washed, 3×2 ml, with 5%methanol in water. The chloroform was then dried over anhydrous sodiumsulfate and the sample was applied to the top of a silica gel column(1×5 cm). The column was eluted with chloroform (5 ml), 5% methanol inchloroform (5 ml), 10% methanol in chloroform (5 ml), and finally 20%methanol in chloroform until all of the product was eluted. Thefractions containing product were pooled and solvent removed on a rotaryevaporator to give the 28 mg of maleimide-PE as white, oily residue. Thematerial was estimated to be greater then 95% pure by thin layerchromatography on silica gel chloroform-methanolacetic acid (60:20:3)and was used without further purification.

DOPG/DOPC sonicated vesicles containing maleimide-PE were prepared bydissolving 16 mg DOPG, 24 mg DOPC and 0.4 mg maleimide-PE in chloroformand removing the solvent by drying under a stream of nitrogen gas. Thisdried film was placed under vacuum overnight to remove traces ofsolvent. The film was then suspended in 0.8 ml water with vigorousshaking and sonicated in a bath type sonicator for 30 minutes untilclear producing DOTMA/DOPC SUV's at a concentration of 50 mg/ml.

The pUC8 plasmid (obtained from BRL, Bethesda Research Laboratory,Gaithersburg, Md., 20877) was grown in E. coli, isolated and purified byprocedures commonly known in the art and stored frozen in water at aconcentration of 1.0 mg/ml.

10 μl of pUC8 plasmid was added to 100 μl of the DOTMA/DOPC vesicles andthoroughly mixed by repeated passage of the sample through a 100 μlHamilton syringe; then a 580 μl aliquot of DOPG/DOPC vesicles containingmaleimide-PE was added to the mixture and thoroughly mixed by repeatedpassage through a 500 μl Hamilton syringe.

The lectin, concanavalin A, was coupled to liposomes through themaleimide-PE moiety by a modification of published procedures (Martin FT, Hubbell W L and Papahadjopoulos D, Biochemistry, 20 4229 (1981);Leserman L D, Machy P and Barbet J, in Liposome Technology, Vol III, ed.Gregoriadis G, CRC Press, Inc, Boca Raton, (1984)). 10 mg ofconcanavalin A in 3 ml PBS, pH 7.0 was reacted withS-acetyl-mercaptosuccinic anhydride (SAMSA; dissolved indimethylformamide at 25 mM). Enough SAMSA was added to give a 50 foldmolar excess of SAMSA over protein and the mixture was incubated at roomtemperature for 1 hour. The reaction mixture was dialyzed against 2×500ml 0.1 M sodium phosphate/0.1 M sodium chloride/5 mM EDTA, pH 7.0. Thedialyzate (approx. 3 ml) was reacted with 0.3 ml 1 M hydroxylaminesolution pH 7.5 for one hour at room temperature. Excess reagent wasremoved on a sephadex G-50 column (1×24 cm) equilibrated with 0.1 Msodium phosphate/0.1 M sodium chloride/5 mM BDTA/10 mMalpha-methylmannoside, pH 7, and maintained under an argon atmosphere.Free sulfhydryl per concanavalin A molecule was determined with5,5′-dithiobis(2-nitro-benzoic acid), Ellman's reagent, and calculatedto be 4 per protein molecule.

Conjugation of the sulfhydryl modified concanavalin A to themaleimide-PE containing, double coated, DNA/DOTMA lipid complexes wasachieved by mixing 0.2 ml of the double coated DNA complexes containingmaleimide-PE, with 2 ml of the concanavalin A (5.5 mg) and the vesselwas capped under an argon atmosphere. The mixture was stirred at 4degrees for three days and then passed through a sepharose 4B column(1.6×73 cm) equilibrated with 50 mM tris/50 mM NaCl/0.05% sodiumazide/10 mM alpha-methylmannoside pH 7.0. The DNA/lipid complexeseluting in the void volume were pooled and dialyzed to removealpha-methylmannoside. The dialyzed vesicles containing the conjugatedconcanavalin A were applied to tissue culture cells and observed byfluorescence microscopy.

B16/F10 tissue culture cells were seeded onto microscope slidescontaining 22×22 mm removable wells. After 24 hours the cells werewashed with PBS and 1 ml of fresh PBS was added to each well. To half ofthe wells was added enough alpha-methylmannoside to give 5 mM and analiquot of the rhodamine containing, concanavalin A conjugated,DNA/lipid complexes was added to each of the wells. The cells wereincubated with the complexes for 1 hour at 37 degrees, washed with PBSand observed by fluorescence microscopy. The cells containingalpha-methylmannoside had significantly less fluorescence on theirsurface then the cells without the sugar.

This result indicates that DNA/DOTMA liposome complexes can be preparedwhich bind specifically to cells.

EXAMPLE 12 Synalar Efficacy in VC Screen

This Example demonstrates the efficacy of a DOTMA liposome solubilizingvehicle. The following results were obtained using the formulationdescribed in Example 4, part 2.

Adult male and female volunteers were selected who did not receive anysteroids and who did not participate in a study using steroids for atleast four weeks prior to testing. The method was based on theStoughton-McKenzie vasoconstriction assay for corticosteroidformulations. All preparations were placed in identical containers,coded and assigned by random tables to individual test sites. Theforearms of the volunteers were prepared by gentle washing and drying.Strips of double adhesive coated BLENDERM(Registered trademark) tapewith 7×7 mm prepunched squares were applied to both forearms, so thateach forearm had 32 application sites. A dose of the assignedformulation was applied to the skin in each square and spread evenly. Aprotective cage was placed over the sites on one arm for “open”application. On the other arm, the remaining backing from the Blendermstrips was removed and the application sites were occluded with SARANWRAP(Register Trademark). After six hours of exposure of the skin to thecorticosteroid preparations, all the tapes were removed and the armswere washed. At 8, 24 and 32 hours after the time of application of theformulations two experienced observers independently scored the presenceor absence of vasoconstriction and the degree of blanching on a scale of0 to 3 under standard lighting conditions.

ASSAY SCORE (# of Sites Responding) Treatment Occluded Assay Open Assay .01% Commercial Solution 100 147  .01% Commercial Cream 210 112  .1%DOTMA Solution 159 150  .03% DOTMA Solution 189 138  .01% DOTMA Solution178 125 .003% DOTMA Solution 175 78 .001% DOTMA Solution 130 60

The results of this experiment demonstrate that the DOTMAliposome/steroid formulation, which is prepared without the use ofsolvents or detergents, is as effective as the conventionalformulations, which use potentially irritating solvents and detergents.

EXAMPLE 13 DOTMA Penetration into Mouse Stratum Corneum

This Example demonstrates the principle that DOTMA liposomes can be usedto introduce drugs into and through the stratum corneum.

Sonicated liposomes in water were prepared containing 14 mM DOPC, 6 mMDOTMA and 0.2 mM rhodamine phosphatidylethanolamine. 0.01 ml of thispreparation was applied to the mouse ear. After 30 min the mouse wassacrificed, the ear was dissected, prepared for cryostat sectioning andexamined by fluorescent microscopy after sectioning. Fluorescentrhodamine-PE was observed to penetrate into the stratum corneum whenDOTMA liposomes were used. When similar sonicated liposomes which didnot contain DOTMA, were applied to the ear, the extent and pattern ofobserved fluorescence was very different. Only staining on the skinsurface was observed; no penetration of fluorescence into the stratumcorneum was detected. This lack of penetration was also observed whenthe dye was dissolved in either acetone or ethanol.

This result indicates that DOTMA liposomes can facilitate stratumcorneum penetration of the rhodamine lipid, or a suitable biologicallyactive medicament such as a lipophilic drug, peptides or proteins andthe like.

EXAMPLE 14 Efficacy of DOTMA Vesicles as an Adjuvant

This Example demonstrates efficacy of DOTMA as an antigen carrier insubunit vaccines. This Example uses the formulation of Example 3, part7.

Groups of 3 guinea pigs were given a single injection of infectiousbovine rhinotracheitis viral antigen, derived from the membraneglycoprotein fraction of virus infected cells, in complete Freund'sadjuvant at 100 mg antigen per guinea pig, or with 150 mg antigen inDOTMA/DSPC multilamellar vesicles (10 mg lipid/guinea pig) with orwithout 0.1 mg/guinea pig6-O-stearoyl-N-acetylmuramyl-L-α-aminobutyryl-D-isoglutamine(6-O-stearoyl[Abu¹] MDP). The animals were bled at 0, 2 and 4 weeksafter injection and the viral neutralizing antibody (reciprocal ofendpoint dilution) in the serum was titered.

SERUM NEUTRALIZING ANTIBODY TITER Treatment 0 Week 2 Week 4 WeekFreund's Adjuvant <5 128 125 DOTMA liposomes <5 130 130 DOTMAliposomes + <5 115 129 6-O-Stearoyl-[Abu¹]-MDP

These results demonstrate that the DOTMA liposomes are as effective asan antigen carrier as is complete Freund's adjuvant.

EXAMPLE 15 Use of DOTMA Vesicles to Achieve Local Skin Penetration ofInterferon

This example demonstrates that DOTMA vesicles can be used to introducelarge drugs, e.g. ,proteins, into the skin from a topical application.Sonicated liposomes containing the lipids as indicated below, at 30mg/ml lipid and Azone® at 2:1 (lipid:Azone®) weight ratio or at 30 mMlipid (2:1 molar ratio, BISHOP/DOPE) were prepared in human or murineinterferon-β solution (in water). Liposome-protein complex were appliedtopically to skin of hairless mice (0.01 ml per spot, 1spot/sample/mouse, 2-3 mice/time point). ¹²⁵I-human β-interferon wasincluded in some preparations of human β-interferon to allow monitoringof uptake of interferon. Mice were sacrificed after 0, 2, 5, and 6hours, and the skin area to which the liposome had been applied waswashed with cotton swabs, excised, and the radioactivity of skin andcotton swab was quantitated in a γ-counter. The following results wereobtained.

Liposome % ¹²⁵I-Interferon Taken Up by Skin Composition (Time, Hr)^(a)with ¹²⁵I-Interferon 0 2 4 6 1. None (free IFN) 0.10 1.19 ± 0.04 0.50 ±0.38 1.21 ± 0.36 2. DOTMA/Azone ® 0.09 5.53 ± 3.89 8.03 ± 3.00 4.93 ±3.44 3. BISHOP/Azone ® 0.15 2.14 ± 1.28 2.51 ± 2.41 0.71 ± 0.52 4.BISHOP/DOPE^(b) 0.16 0.68 ± 0.11 1.25 ± 0.47 0.65 ± 0.32 ^(a)Mean ± S.D.of two mice; 0 hr sample, one mouse per sample. ^(b)BISHOP =2,3-Bis(hexadecyloxy)propyl-N,N,N-trimethylammonium chloride a.k.a.,N-(2,3-dihexadecyloxy)-prop-1-yl-N,N,N-trimethyl-ammonium chloride; DOPE= dioleoyl-L-α-phosphatidylethanolamine.

These results demonstrate that liposomes containing positively-chargedlipids of Formula I promoted skin uptake of interferon.

To further assess this skin penetration by interferon, another test wasconducted in which murine β-interferon-liposome complexes were incubatedwith mouse skin as above for 4 hours, mice were sacrificed and skin waswashed and excised. Skin sections were frozen and thin sections preparedwith a cyrostat. These sections were then incubated with rabbit IgGantibodies to murine interferon-β and washed. Second antibodies,FITC-labeled, against rabbit IgG were then added, incubated for anadditional 30 minutes at 37° C., washed, and tissue samples wereanalyzed under the fluorescent microscope. Tissue sections from miceoriginally treated with interferon complexed with Dotma:Azone® liposomesshowed significant penetration of interferon through the stratumcorneum. By contrast, tissue sections from mice originally treated withfree interferon did not show any appreciable skin penetration ofinterferon.

EXAMPLE 16 Double Stranded RNA Entrapment Using the Methodology forDouble Coated Complexes

Commercially available (Beohringer-Mannheim) double stranded RNA (dsRNA)(poly IC) is very heterogenous with respect to size. This can bedemonstrated by passing an aliquot of dsRNA disolved in isotonic saline(0.9% sodium chloride, 50 mM HEPES, pH 7.0) over a gel filtration column(Sepharose CL-2B) equilibrated in the same buffer. Experiment 1indicates that the double stranded RNA migrates in a very broad peakranging between the void volume and the included volume of this column.Electrophoretic agarose gels likewise indicated a very heterogenous sizedistribution ranging between 100 and 3500 base pairs in length. However,we determined that sonication (2 hours, 15 degrees centigrade, in asealed vial, using the inverted cup of a Branson 350 cell disrupter atthe maximum setting) could reduce the size of the dsRNA to about 130base pairs. The gel filtration data for the sonicated dsRNA is alsoshown on Experiment 1. These results clearly indicate that the size ofthe dsRNA was reduced and the sample reached a more uniform sizedistribution than the starting material. Gel electrophoresis of thesample confirmed this conclusion and revealed that the resulting samplemigrated with a size of about 130 base pairs compared to double strandedDNA.

A second experiment was done in order to determine whether any of dsRNAcould be stably entrapped using a double coating procedure similar tothe one described in Example 9. The sonicated dsRNA was mixed in a 1 to10 weight ratio with positively charged DOTMA/DOPE liposomes by theusual methods described in example 8. The resulting solution containsDOTMA liposome/dsRNA complexes. This solution was mixed with a secondsolution of negatively charged liposomes (DOPG/DOPC) (1:1) so as toproduce the double-coated dsRNA liposome complexes. The quantity ofnegatively charged liposomes exceeded the positively charged DOTMAliposomes by a factor of 10 so that the resulting complexes contained anet negative charge. These negatively charged dsRNA complexes werepassed over the same gel filtration column. The data shown in Experiment2 indicate that the free negatively charged liposomes (monitored byusing a trace amount of ¹⁴C labeled phospholipid during the liposomeformulation) before complexation with dsRNA, migrate well into the gelfiltration column. However, the same negatively charged liposomes, aftercomplexation with dsRNA complexes, give rise to a different gelfiltration pattern; approximately 30% of the lipid appears in the voidvolume, indicating that this fraction of the lipid is involved in arelatively large complex with the dsRNA. A similar fraction (i.e., 30%)of the dsRNA (monitored by using an assay employing ethidium bromide) isalso involved in the high molecular weight complex, which is consistentwith this conclusion. The void volume fractions containing the doublecoated negatively charged dsRNA complexes were pooled and rerun over thegel filtration column and all of the applied dsRNA and radioactive lipidmigrated together in the void volume (data not shown). This resultindicted that the double coated complexes were stable.

This example illustrates a convenient method, by simple mixing ofpremade liposome solutions, for the stable entrapment of a highpercentage of polynucleotide into negatively charged complexes.

Experiment 2, Example 16: Elution Profile of dsRNA and Experiment 1,Example 16: Lipid Complexes from a Elution Profile of dsRNA SepharoseCL-2B Column from a Sepharose CL-23 Column Percent Recovery PercentRecovery Double Fraction Original Sonicated Free Coated Complexes NumberRNA RNA [¹⁴C]DOPG/DOPC RNA [¹⁴C]DOPG/DOPC 1 .564 .065 .540 .041 2 .108.044 .540 .030 3 .075 .564 .073 .216 .041 4 .077 .513 .041 5 .075 .564.052 .540 .041 6 .075 .564 .089 .540 .038 7 .085 .540 .046 8 .075 .564.081 .216 .024 9 .345 .564 .134 .405 .212 10 2.073 .564 .955 .945 3.11211 2.829 .580 1.166 7.297 5.272 12 3.207 .590 1.227 9.459 3.250 13 3.639.661 1.296 5.540 1.988 14 4.341 .800 1.439 3.243 1.446 15 4.665 1.0481.727 1.891 1.446 16 5.097 1.451 1.882 1.351 1.663 17 5.745 2.177 2.1461.486 1.826 18 7.041 3.048 2.471 1.621 2.170 19 6.987 4.322 2.951 1.7562.537 20 7.365 5.741 3.841 1.891 3.008 21 7.851 7.354 4.886 2.162 3.54822 7.905 8.870 6.345 2.432 4.669 23 9.935 8.138 2.972 6.033 24 6.55510.758 10.060 3.783 8.179 25 5.691 10.774 12.077 4.729 10.245 26 4.28710.435 11.865 5.945 11.278 27 3.909 9.290 9.674 6.891 10.110 28 2.3977.629 7.536 7.297 8.292 29 1.749 6.300 4.890 7.837 5.578 30 .885 4.2902.817 6.486 3.247 31 .669 2.870 1.300 5.135 1.716 32 .237 2.000 .6543.378 .829 33 .291 1.322 .422 2.027 .432 34 .247 1.297 .201 35 .1291.032 .195 .810 .151 36 .138 .945 .107 37 .121 .675 .107 38 .105 .000.099

EXAMPLE 17 Use of DOTMA to Increase Interferon Inducing Activity ofdsRNA in Tissue Culture Cells

Interferon treatment of various cells including both mouse and humancells results in induction of an enzyme, 2-5A synthetase, which whenactivated by double stranded RNA produces 2-5A. 2-5A is a polymer of ATPthat contains unique 2′-5′ linkages rather than the standard 3′-5′linkages. 2-5A is effectively a communication molecule which activates alatent 2′-5′ endonuclease resulting in degradation of cellular mRNA. Inthe absence of double stranded mRNA the 2-5A synthetase remainsinactive, and no 2-5A is produced. In the presence of double strandedRNA, 2-5A is produced and can be produced in sufficient concentrationsto degrade the majority of the cellular RNA, including ribosomal RNA,resulting in cell death. In this respect, this system can be used ascytotoxic assay to analyze the capacity of a compound to introducedouble stranded RNA into the cytoplasm of a cell. The double strandedRNA commonly used is poly IC, a commercially available compound which isused experimentally and classically to induce interferon by methods wellknown to those familiar in the art. Poly IC will not penetrate cells andcause cytotoxic effects alone, but requires a vehicle to help carry itacross the cell membrane. The most common agent used for this purpose isDEAE dextran in experimental cell culture systems and a complex ofcarboxymethyl cellulose and polylysine in clinical use. Both vehicles,however, are inefficient at introducing poly IC into the cell so thatonly a fraction of the cells are killed in an interferon inducedcytotoxic assay. In addition DEAE dextran is itself toxic so that atconcentrations where it acts effectively, it contributes significantlyto the cellular cytotoxicity observed in experimental systems. Aneffective vehicle would allow high efficiency introduction of poly ICwithout contributing to cytotoxicity itself. The DOTMA lipid is such avehicle. In experimental tests the frequency of surviving cells whichhave poly IC introduced by the DOTMA lipid is 1×10⁻⁶. The DOTMA, whenused under standard conditions, effectively introduces sufficient dsRNAinto essentially every cell on a 10 cm² petri dish. The occasionalsurviving cells are not cells which merely have not received dsRNA, butrather have been mutants which are resistant to the cytotoxicity ofdsRNA. These results indicate that the DOTMA lipid is an ideal vehiclefor introducing double stranded RNA and other nucleic acids into cells.

TABLE I^(a) Treatment Cell Survival Control 100% DOTMA/DOPE 100%Liposomes (50 g/10 ml) Poly IC 100% (1 g/10 ml) Liposomes + 1 × 10⁻⁵%Poly IC ^(a)1 million mouse L-cells were seaded onto 10 cm² culturedishes in Eagle's minimal essential media (Gibco) containing 10% fetalcalf serum. After 24 hours the cells were challanged with the treatmentsindicated in Table I. After an additional 24 hrs the cells were washedand stained with trypan blue to determine viability.

EXAMPLE 18 Comprehensive DNA Transfection Method

1. Introduction

A large body of work has demonstrated that under appropriate conditions,eukaryotic cells can take up exogenous DNA and that a portion of thisDNA becomes localized in the nucleus. This phenomenon has been exploitedin order to obtain both transient and stable expression of variousgenes. However, due in part to the size and charge of DNA and to themultitude of enzymatic and membrane barriers imposed by the cell, thespontaneous entry of intact DNA into the cell and its subsequentexpression in the nucleus is a very inefficient process. For thisreason, a wide variety of methods have been developed in order tofacilitate this process. These methods include the use of polycations,calcium phosphate, liposome fusion, retroviruses, microinjection,electroporation, and protoplast fusion. However, all of these methodssuffer from one or more problems related to either cellular toxicity,poor reproducibility, inconvenience or inefficiency of DNA delivery.

We have recently synthesized a cationic lipid which forms liposomes. Weshow here that these liposomes interact with DNA spontaneously, fusewith tissue culture cells and facilitate the delivery of functional DNAinto the cell. The technique is simple, highly reproducible and moreefficient than some other commonly used procedures.

2. Methods

(A) Cells and Media

The cell line, COS-7 (ATCC CRL 1651) is a derivative of the simiankidney cell line CVI (ATCC CCL70) transformed with a mutant of SV40. ψ2is a murine fibroblast cell line that stably expresses a packagingdeficient retrovirus; it is used, most often, for the production ofretroviral vector stocks. MSN610.2 is a glucocorticoid receptordeficient subclone of the mouse mammary tumor virus (MMTV)-infected, ratHTC derived cell line MSC.1. JZ.1 is a HTC cell line containing oneintegrated copy of MMTV. L-tkcells (ATCC CCL 1.3) are derived frommurine L-cells and are thymidine kinase deficient. The TA1 cell line isderived from the murine fibroblast cell line 10%½. All cells were grownon plastic tissue culture plates in DMEM+10% foetal calf serum (f.c.s.)and in a 10% CO₂, 37° incubator except TA1 cells which were grown inBMEM+10% f.c.s. in a 5% CO₂, 37° incubator.

(B) DOTMA Synthesis and Liposome Preparation

DOTMA (N-(l-(2,3,-Dioleyloxy)propyl)-N,N,N-trimethylammonium chloride)was prepared as outlined Reaction Scheme V. A mixture of3-(dimethylamino)-1,2-propanediol (Aldrich Chemical Co., 1.19 g, 10mmol), potassium tert-butoxide (3.36 g, 30 mmol) and oleylp-toluenesulfonate (12.7 g, 30 mmol) in xylenes (50 ml) was stirred atroom temperature and reduced pressure (30 Torr) for 30 min, and was thenheated to 50° C. with stirring for an additional 15 min. The reactionvessel was purged with nitrogen, and the mixture was heated to reflux(approximately 140° C.) for 3 hrs. After cooling, the reaction mixturewas diluted with hexane (100 ml), and the resulting solution wasextracted with water (2×50 ml). The organic layer was concentrated, andthe residue was chromatographed over silica gel by elution with amixture of hexanes and ether (1:2) to afford the intermediate2,3-dioleyloxy-1-(dimethylamino)-propane as a colorless oil.Quaternization was carried out by condensation of methyl chloride (50ml) into a Parr pressure apparatus cooled to −78° C. containing thiscompound (10 g). The sealed vessel was heated behind a safety shield at70° C. for 48 h. After cooling to 0° C., the reaction vessel was opened,and the excess methyl chloride was allowed to evaporate under a streamof nitrogen in a well-ventilated hood. The crude residue wasrecrystallized from acetonitrile to afford DOTMA as an off-white solid,mp. 35-38° C. Full details of the synthesis of DOTMA and analogs will bepublished elsewhere (Gadek and Felgner, in preparation).

A solution of PR (10 mg) and DOTMA (10 mg) in chloroform (1 ml) wasevaporated to dryness under a stream of nitrogen gas and residualsolvent was removed under vacuum overnight. Liposomes were prepared byresuspending the lipids in deionized H₂O (2 ml) and sonicating toclarity in a closed vial. Sterile preparations of liposomes are stablefor at least 6 months at 4° C.

(C) Transfection of Cells using DOTMA (Lipofection)

Plasmid DNAs were purified by the method of Birnboim and Doly withsubsequent removal of high Mr RNA by precipitation with 2.5M LiCl andbanding on CsCl gradients. Plasmids pSV2CAT, pSV2neo. pZipneoSVX is aretroviral vector which encodes a neomycin resistance gene. pZipC5a andpMSGc5a are derived from pZipneoSVX and pMSG (Pharmacia) respectively bythe insertion, in the anti-sense orientation, of a cDNA isolatedrecently from adipogenic cells (clone 5 from reference 17).

The details of individual transfections are given in the resultssection. A general protocol for transfections is given below.

(a) Formation of the lipid-DNA complexBoth DNA and lipid are diluted to1.5 ml each with HBS (150 mM NaCl, 20 mM Hepes pH 7.4) and then mixed.The DNA lipid complexes form immediately. A typical transfection woulduse 1 to 10 μg DNA and 100 μg of total lipid (DOTMA:PE, 1:1).

(b) Treatment of cells—Just confluent 100 mm tissue culture plates ofcells are washed 2× with 5 ml of HBS and 3 ml of the DNA-lipid solutionis added. The cells are incubated for 3-5 hours at 37° and then 10 ml ofDMEM+10% fcs is added. After incubation for 16 hours at 37° the mediumis replaced with 10 ml fresh medium and cells are harvested by scraping2-3 days later. Cell extracts were prepared and CAT assays performed aspreviously described. For stable transfections 50% confluent cells weretreated as above except that 2 days after transfection cells werepassaged and grown in selective medium for either neomycin resistance(0.4 mg/ml G418) or E. coli XGPRT expression.

(D) Transfection by Calcium Phosphate Preciptiation and by DEAE-Dextran

Cells were transfected with calcium phosphate precipitated DNA asdescribed with the addition of a glycerol shock. Similarly cells weretransfected with the DEAE-dextran method as described.

(E) Staining of Cells with Rhodamine-Conjugated Lipid

A solution of DOTMA (10 mg), rhodamine-PE (Avanti Polar Lipids, Inc.;0.2 mg) and either PR (10 mg) or PC (10 mg) in chloroform (1 ml) wasevaporated to dryness under a stream of nitrogen gas and residualsolvent was removed under vacuum overnight. Liposomes were prepared byresuspending the lipids in deionized H₂O and sonicating to clarity in aclosed vial. Fluorescent lipid-DNA complexes were prepared from theseliposomes by mixing 0.5 ml of liposomes (0.1 mg/ml total lipid in HBS)and 0.5 ml of pSVCAT DNA (0.02 mg/ml in HBS). The complexes (5 μg) wereadded to mouse L-cells (1×10⁵) which had been seeded onto microscopeslides containing 2 cm×2 cm wells. After a four hour incubation thecells were washed with HBS and examined by epifluorescence microscopy.

3. Results

(A) Formation of DNA-Lipid Complexes

Cationic lipid vesicles might be expected to have the desirableproperties of both cationic mediators of DNA transfection (e.g.,spontaneous complex formation with DNA and the cell surface) and ofliposome mediated transfection (rapid fusion and uptake of the DNA).However there are no widely available cationic, bilayer-forming lipidswhich give rise to physically stable liposomes. We, therefore, designedand synthesized a cationic lipid, DOTMA (Reaction Scheme V) which eitheralone or in combination with neutral phospholipids, spontaneously formsmultilamellar liposomes which can be sonicated to form small unilamellarvesicles (not shown). The rectangular array of the parallel alkyl chainsmay be a significant factor contributing to the formation and stabilityof DOTMA bilayers as has been shown for other lipids. Thecharacterization of these liposomes will be presented in detailelsewhere.

DNA interacts spontaneously with solutions of DOTMA to form lipid-DNAcomplexes. This complex formation is due presumably to ionicinteractions between the positively charged group on the DOTMA moleculeand the negatively charged phosphate groups on the DNA. Complexformation was examined using a sucrose density gradient (not shown). Inthe absence of lipid, DNA migrated to the bottom of the gradient whilelipid, in the absence of DNA, floated at the top. When lipid was mixedwith DNA at a ratio of 5:1 (wt:wt) all the DNA migrated with the lipid.The association of 100% of the DNA with DOTMA after gentle mixingcontrasts with conventional liposome encapsulation procedures whichusually entrap less than 10% of the DNA, require an additionalpurification step to remove unencapsulated DNA or involve potentiallydestructive conditions such as vigorous agitation or sonication.

(C) Fusion of DNA-Lipid Complexes with Cells

We speculated that the positively charged lipid, DOTMA, would not onlyinteract with DNA to form a complex, but would also cause the complex tobind to tissue culture cells and possibly fuse with the plasma membrane.Incorporation of rhodamine conjugated phosphatidyl ethanolamine into theDNA-lipid complex allows one to follow the fate of the complex as itinteracts with tissue culture cells. Fluorescence microscopy revealedthat the complexes fuse with the cell membrane and diffuse throughoutthe intracellular membranes. Furthermore the intensity of cellassociated fluorescense increased with time reaching a maximum after 4hrs when virtually all the cells were fluorescently labelled. Ourstandard transfection cocktail contains DOTMA and a neutral lipid,phosphatidylethanolamine (DOPE), in a 1:1 (wt:wt) ratio (see materialsand methods). The fusogenic capabilities of the complex can becontrolled to a certain extent by the choice of the neutral lipid usedto form the complex. The substitution of phosphatidylcholine for PR forinstance, inhibits fusion of the complex with the cell membrane and apunctate, surface associated fluorescence is seen on tissue culturecells. These results are what one might predict from the known fusogenicproperties of DOPE.

(D) Optimization of the Transfection Protocol

Cells transfected with the plasmid pSV2CAT express CAT enzyme activitythat can be measured in cell extracts 2 days after transfection. Thelipid transfection technique was optimized for this kind of transienttransfection assay using two monkey kidney cell lines CV1 and COS-7.COS-7 cells are often used for transient transfection assays as theyproduce SV40 T-antigen which allows replication of plasmids, such aspSV2CAT, containing an SV40 origin of replication. CV1 is the parentalcell line which does not produce T-antigen and in which pSV2CAT cannotreplicate.

(i) Concentration of DNA

Only 1 μg of pSV2CAT is required for the optimum transfection of COS-7cells. Furthermore, transfection efficiency is relatively insensitive toa broad range of DNA concentrations (FIG. 1A); only a two folddifference in CAT activity is observed when 0.2 to 40 μg of pSV2CAT areused. In CV1 cells, maximal expression of CAT activity is achieved with10 μg of DNA and there is a significant decrease with lowerconcentrations. In both CV1 and COS-7 cells very high DNA levels havesome inhibitory effect. The difference in the dependence on DNAconcentration between CV1 and COS-7 cells probably lies with the abilityof COS-7 cells to replicate pSV2CAT to a high copy number. PresumablyCOS-7 cells are less dependent on the amount of DNA taken up by eachcell than on the percentage of cells transfected. CVI cells on the otherhand would be dependent on both the amount taken up by each cell and onthe transfection frequency.

(ii) Concentration of Lipid

Increasing concentrations of lipid improve transfection of both CV1 andCOS-7 cells (FIG. 1B). However, the lipid is toxic to these cells athigh levels (above 100 μg) and though an increase in specific activityof the cell extract can be obtained, the yield of enzyme activitydecreases due to cell death. We have transfected a number of cell typeswhich grow as monolayers and in every case satisfactory transfection wasobtained using sub-toxic levels of lipid.

(iii) Time of Incubation with DOTMA:DNA Complexes

In standard transfections cells were incubated with the lipid-DNAmixture for 3 hrs. Growth medium was then added to the cells and after afurther 16 hrs this mixture was replaced with fresh medium. The lengthof the initial incubation period was varied and the results arepresented in FIG. 1C. The addition of growth medium after 5 minutes oftreatment of CV1 cells with the DNA-lipid mixture almost completelyinhibits transfection. As the length of the treatment with the DNA-lipidcomplex increases, so does the transfection efficiency. An optimumtransfection time of 5 hrs, was determined for both CV1 and COS cellsand incubations of greater than 8 hr with lipid resulted in unacceptablelevels of toxicity; this may vary however in other cell lines. Evenafter 3 hrs of treatment, however, replacement with fresh medium yieldedonly 7% of the CAT activity obtained when medium was added to cells inthe continuing presence of the DNA:lipid complex (FIG. 1C). We have alsoobserved that transfection of mouse L-cells with DOTMA is inhibited byserum-containing growth medium. In particular, when serum was presentfrom the outset, CAT activity was reduced to less than 5% of control(data not shown). COS-7 cells, in contrast to CV1 and L-cells cells,appear partially refractory to the presence of serum-containing growthmedium (FIG. 1C). Furthermore, washing the cells after 3 hrs oftreatment with the DNA-lipid complex does not reduce the transfectionefficiency, as measured by CAT activity, when compared to the additionof growth medium after 3 hrs. We suspect that the relative insensitivityof COS cells again reflects their ability to replicate pSV2CAT to highcopy number.

(D) Lipofection vs DEAE-dextran and calcium phosphate for transient andstable transfection of cells

The ability of DOTMA to facilitate DNA uptake and functional expressionwas compared with the commonly used mediators of transfection,DEAE-dextran and calcium phosphate. In CV-1 and COS 7 cells, DOTMAyielded a 6-11 fold increase in CAT activity relative to transienttransfection with DEAE-dextran (Table 1). A variety of cell lines,including the rat hepatoma HTC cell line, seem to be highly refractoryto transient transfection with DEAE-dextran. In contrast, DOTMAcomplexes of pSV2CAT yielded reproducible transfections of JZ.1 cells (asubclone of HTC cells) exhibiting at least an 80-fold enhancement overthe DEAE-dextran procedure.

DOTMA is not only useful in transient assays but can also be used tofacilitate the stable transformation of cells. We have analyzed thefrequency of stable transfection of various cell lines using DOTMA andcompared it to the frequency obtained using calcium phosphateprecipitation of DNA (table 2). In four different cell lines, DOTMAyielded from 6 to greater than 80 times as many stable transformantsusing either neomycin (G418) resistance or E. coli XGPRT expression asthe selectable markers.

3. Discussion

We have described the use of liposomes containing the cationic lipidDOTMA to facilitate the functional delivery of DNA into cells. Thespontaneous formation of DNA-DOTMA complexes which are effective in DNAtransfection suggests to us that a single plasmid is surrounded bysufficient cationic lipid to completely reverse the charge of the DNAand provide a net positively charged complex that would allowassociation with the negatively charged surface of the cell. Thetechnique works well for both stable and transient expression of theintroduced DNA and with several cell types we have studied it is moreefficient than either DEAE-dextran or calcium phosphate precipitation.

The quantity of DNA required for an optimum signal in transienttransfections varies with cell type. COS-7 cells which replicate pSV2CATto a high copy number require only 1 μg of DNA while CV1 cells require10 μg. In both cases DNA levels above the optimum are inhibitoryalthough this effect is small. Indeed with COS-7 cells varying the DNAlevel from 0.2 to 40 μg gives a transfection signal which is never lessthan 50% of the maximum obtained. This, together with the observationthat DOTMA-mediated transfection is effective for impure DNApreparations such as those obtained from ‘mini-preps’ would allow therapid screening of new constructions by transfection. The smallestquantity of DNA required for a detectable signal depends to a largeextent on the DNA and detection system used. With pSV2CAT in COS-7 cells1 ng of DNA (without carrier) gives rise to an easily detectable CATsignal. Substitution of the SV40 early promoter with the Rous sarcomaviral promoter (pRSVCAT) allows CAT enzyme levels to be detected with aslittle as 0.1 ng of DNA (Northrop, unpublished). Moreover, with bothpSV2CAT and pRSVCAT the addition of carrier DNA does not adverselyaffect the transfection signal obtained.

The concentration of lipid used in a transfection depends on the celltype. We have been able to obtain satisfactory transfection, both stableand transient with DOTMA:PE levels between 50 and 100 μg. Increasing thelipid concentration above these levels may increase the specificactivity of the cell extract but significant toxicity also occurs. Thetoxicity varies with the type of cell, the duration of exposure toDOTMA, and also with the density of the cell culture; dense cultures aremore resistant to the toxic effects of the lipid than less densecultures. Although high levels of lipid are toxic, in our hands itappears to be less toxic than the concentration of DEAE-dextran which isrequired for optimum transfection of most cell types. Based on ourexperience it is best to optimize the various parameters described abovefor each cell line.

The exact composition of the DOTMA containing liposomes can be variedsince pure DOTMA liposomes are almost as effective as DOTMA:PE (1:1)liposomes. If, however, the PE containing liposomes are formulated witha negatively charged lipid, such as phosphatidylglycerol, rather thanwith DOTMA, transfection is completely abolished. Surprisingly, twocommercially available cationic lipids which bear significant structuralsimilarity to DOTMA, stearylamine and dioctadecyl dimethyl ammoniumbromide have shown little efficacy as mediators of DNA transfection withmouse L-cells (Felgner, unpublished). The properties of DOTMA containingliposomes we have described here suggest that this method may also haveutility for introducing large DNA molecules, oligonucleotides, and RNAsinto mammalian cells.

TABLE 1 Transient transfections: lipid compared with DEAE-dextran DEAE-Lipid dextran Time DNA CAT sp. act. Cell Line (μg) (mg/ml) (hrs) (μg) (%max) JZ.1 100 3 5 19 100 3 25 100 150 3 25 80 0.25 5 5 <1 0.25 5 25 10.25 16 25 <1 0.50 5 25 1 CV-1 100 5 10 100 0.25 5 10 8.9 COS-7 100 5 1100 0.25 5 1 7 0.25 5 10 16 0.50 5 1 7 0.50 5 10 11 Transfections werecarried out as described in the Methods; lipid corresponds to DOTMA:PE(1:1). Each section of the table represents an independent experimentand in each case the transfection that yielded the highest level of CATactivity was set at 100%. All transfections were performed in duplicateand CAT assays from each were performed in duplicate. (+) denotes theaverage results from two completely independent experiments.

TABLE 2 Comparisons of lipid and calcium phosphate stable transfectionsFrequency × Cell Line Plasmid Transfection 10⁵ L-TK pSV2neo CaCl₂ 3Lipid 45 Ψ2 pZIPSVX CaCl₂ 0.6 Lipid >49 Ψ2 pZIPC5a CaCl₂ 1.8 Lipid >68MSN610.2 pSV2neo CaCl₂ 1.3 Lipid 8.2 TA1 pSV2neo CaCl₂ 2 Lipid 14 TA1pZIPSVX CaCl₂ 0.7 Lipid 17 TA1 pMSGC5a CaCl₂ 1.3 Lipid 19 Cells weretransfected with 7 μg of the indicated plasmid with no carrier DNAexcept for pSV2neo where 1 μg of plasmid with 10 μg of carrier DNA wereused. The transfection frequency is the number of drug resistantcolonies expressed as a fraction of the total number of cells plated.

What is claimed is:
 1. A method for introducing nucleic acids intomammalian cells which method comprises forming liposomes from positivelycharged lipids, contacting the liposomes with the nucleic acids to formnucleic acid-liposome complexes, wherein the nucleic acids are notencapsulated within the aqueous phase of the liposomes, and contactingthe complexes with the mammalian cells under in vivo or in vitroconditions which are conducive to the transfection of the mammaliancells.
 2. A method of claim 1 wherein said nucleic acid is apolynucleotide.
 3. A method of claim 2 wherein the polynucleotide is aDNA or an RNA comprising naturally occurring bases, modified bases, ormixtures thereof.
 4. A method of claim 1 wherein the second contactingstep is performed in vitro.
 5. A method of claim 1 wherein the secondcontacting step is performed in vivo.
 6. A method of claim 1 wherein thepositively charged lipid contains at least one quaternary ammoniumgroup.
 7. A method of claim 1 wherein the nucleic acid-liposome complexis positively charged.
 8. A method for transfecting mammalian cells withpolynucleotides which method comprises forming liposomes from positivelycharged lipids, complexing the liposomes with the polynucleotides toform polynucleotide-liposome complexes, wherein the polynucleotides arenot encapsulated within the aqueous phase of the liposomes, andcontacting the complexes with mammalian cells, under in vivo or in vitroconditions which are conducive to transfection of the cells.
 9. A methodof claim 8 wherein the polynucleotide is a DNA or an RNA comprisingnaturally occurring bases, modified bases, or mixtures thereof.
 10. Amethod of claim 8 wherein the contacting step is performed In vitro. 11.A method of claim 8 wherein the contacting step is performed in vivo.12. A method of claim 8 wherein the positively charged lipid contains atleast one quaternary ammonium group.
 13. A method of claim 8 wherein thepolynucleotide-liposome complex is positively charged.
 14. A method fortransfecting mammalian cells with polynucleotides which method comprisescontacting the mammalian cells with polynucleotide-liposome complexes,under in vivo or in vitro conditions which are conducive totransfection, said liposome formed from positively charged lipids, saidpolynucleotides not encapsulated within the aqueous phase of theliposomes.
 15. A method of claim 14 wherein the polynucleotide is a DNAor an RNA comprising naturally occurring bases, modified bases, ormixtures thereof.
 16. A method of claim 14 wherein the contacting stepis performed in vitro.
 17. A method of claim 14 wherein the contactingstep is performed in vivo.
 18. A method of claim 14 wherein thepositively charged lipid contains at least one quaternary ammoniumgroup.
 19. A method of claim 14 wherein the polynucleotide-liposomecomplex is positively charged.
 20. A cell modified by the process ofclaim 15 comprising native cellular elements, the polynucleotide, andexpression products of the polynucleotide.
 21. A method of claim 1 formodifying the genetic complement of human cells comprising thetransfection of exogenous polynucleotides into the human cells bycontacting the human cells, under in vivo or in vitro conditions whichare conducive to transfection, with polynucleotide-liposome complexes,said polynucleotides not encapsulated within the aqueous phase of theliposomes.
 22. A method of claim 21 wherein the cells are transfected invitro.
 23. A method of claim 21 wherein the cells are transfected invivo.
 24. A method of claim 1 for gene delivery in humans which methodcomprises transfecting exogenous polynucleotides into targeted humancells by contacting the human cells, under in vivo or in vitroconditions which are conducive to transfection, withpolynucleotide-liposome complexes, said polynucleotides not encapsulatedwithin the aqueous phase of the liposomes, and expressing the introducedpolynucleotides.
 25. A method of claim 24 wherein the cells aretransfected in vitro.
 26. A method of claim 24 wherein the cells aretransfected in vivo.